Compositions comprising liposomes composed of whole cell membrane fraction are provided. The liposomes may be attached to, or encapsulate a pharmaceutical agent. Also provided are methods of generating and using these lipospmes....https://www.google.co.il/patents/US20120164214?utm_source=gb-gplus-sharePatent US20120164214 - Liposomal compositions and uses of same

Compositions comprising liposomes composed of whole cell membrane fraction are provided. The liposomes may be attached to, or encapsulate a pharmaceutical agent. Also provided are methods of generating and using these lipospmes.

Images(9)

Claims(27)

1. A composition-of matter comprising a liposome attached to, or encapsulating a pharmaceutical agent, said liposome being composed of a whole cell membrane fraction.

2. The composition-of-matter of claim 1, wherein said cell is a human cell.

3. The composition-of-matter of claim 1, wherein a cell source for said whole cell membrane is selected from the group consisting of a stem cell, a primary cell, a cell-line, a non-tumorigenic cell, a cancer cell and an immune cell.

4. A composition-of matter comprising a liposome composed of a whole cell membrane fraction of a stem cell.

(d) extruding said ruptured membrane and/or ghosts through a matrix of pre-determined porosity.

23. The method of claim 21 further comprising conjugating a synthetic polymer to said liposomes following step (c).

24. A method of encapsulating a pharmaceutical agent in a liposome, the method comprising making the liposomes according to the method of claim 21 and adding the pharmaceutical agent prior to the step of homogenizing.

25. A pharmaceutical composition comprising as an active ingredient the composition-of-matter of claim 1 and a pharmaceutically acceptable carrier.

26. A method of delivering a pharmaceutical agent, the method comprising administering to a subject in need thereof the composition of matter of claim 1, thereby delivering the pharmaceutical agent.

27-28. (canceled)

Description

RELATED APPLICATION/S

[0001]

This application claims the benefit of priority under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/237,306 filed Aug. 27, 2009, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

[0002]

The present invention, in some embodiments thereof, relates to liposomal compositions and uses of same.

[0003]

Liposome based DNA and drug delivery systems have been extensively investigated in the last four decades, and used as a mean to treat various conditions. Liposomal systems allow the efficient entrapment of both hydrophilic and hydrophobic compounds in a well-characterized, biocompatible and non-immunogenic lipid vesicle that can range from nanometers to micrometers in diameter. Liposomes can also be targeted using specific ligands such as protein conjugates or antibodies that bind specific cellular receptors. In cancer therapy, liposomal systems are of the most popular and well-investigated drug carriers. This is mainly due to the enhanced permeability and retention (EPR) effect, which refers to the increased vascular permeability of tumor vessels due to tumor angiogenesis. The EPR effect results in the accumulation of liposomes in the tumor extracellular fluid, which is exploited as a passive targeting mechanism. State of the art technologies in liposomal drug delivery for cancer therapy primarily include drugs that are approved for clinical use (e.g., DaunoXome™, Myocet™, Doxil™, Caelyx™). Several approaches are currently investigated for the targeting of liposomal systems to cancer, which include the binding of targeting moieties to the liposome surface (e.g., antibodies). Synthetic cationic liposomes are the most common vectors for DNA delivery although their cytotoxicity remains a concern irrespective of the preferred route of DNA transfer both in vitro and in vivo. On the other hand, anionic liposomes that better resemble cell-derived liposomes (in term of their electric charge) were also shown to mediate gene transfer, but suffer from poor encapsulation efficiency due to the large size and the negative charge of the uncondensed DNA. Improving encapsulation efficiency and protecting DNA from degradation was achieved by complexation of the DNA with cations or poly-cations that subsequently also significantly improved the transfection efficiencies.

[0004]

In the last decade several studies have revealed that certain primary cells, such as adult mesenchymal stem cells (MSC), adult hematopoietic stem cells (HSC) and endothelial cells, accumulate at tumor microenvironments, when administered to tumor bearing animals. Recent data suggests that isolated membrane fractions of tumor cells appear to contain potent MSC attractants, more so than the cytoplasmic fractions isolated from the same cells. This data implies that the mechanism of MSC targeting to tumor cells is mainly governed by cell-to-cell interactions via the binding of surface molecules found on tumors and MSC. However, cellular response to different soluble factors (i.e., chemokines) secreted by angiogenic blood vessels and tumor cells is suggested to take some part in the MSC homing mechanism as well. The homing mechanism motivated studies on the use of these cells as a targeted delivery vehicle for cancer therapy. In these studies, primary cells were isolated and transduced with different genes of interest, either anti-cancer or reporter genes. The cells were transplanted to tumor bearing animals and their homing to the tumor microenvironment was demonstrated using the expressed reporter proteins. Tumor inhibition was achieved using the expressed anti-cancer proteins.

[0005]

Liposomes, which are derived from the cytoplasmatic membrane of mammalian cells, have been commonly used as a tool in the study of membranes and cellular mechanisms. Cell derived liposomes (CDL or CDLs in plural) have been also investigated as a tool for cancer immunotherapy. In these studies, liposomes were prepared from the membranes of tumor cells and were used as adjuvant to evoke the immune system towards tumor antigens located on the liposome membrane. However, cell derived liposomes have never been produced from stem cells, nor used as a delivery vehicle. Furthermore, no CDL system has ever been developed as a targeting platform.

According to an aspect of some embodiments of the present invention there is provided a composition-of matter comprising a liposome attached to, or encapsulating a pharmaceutical agent, the liposome being composed of a whole cell membrane fraction.

[0009]

According to some embodiments of the invention, the cell is a human cell.

[0010]

According to some embodiments of the invention, a cell source for the whole cell membrane is selected from the group consisting of a stem cell, a primary cell, a cell-line, a non-tumorigenic cell, a cancer cell and an immune cell.

[0011]

According to an aspect of some embodiments of the present invention, there is provided a composition-of matter comprising a liposome composed of a whole cell membrane fraction of a stem cell.

[0012]

According to some embodiments of the invention, the stem cell comprises a human mesenchymal stem cell.

[0013]

According to an aspect of some embodiments of the present invention there is provided a composition-of matter comprising a liposome composed of a whole cell membrane fraction of a primary human cell.

[0014]

According to an aspect of some embodiments of the present invention there is provided a composition-of matter comprising a liposome composed of a whole cell membrane fraction of a non-tumorigenic human cell.

[0015]

According to some embodiments of the invention, the cell membrane is genetically modified to express an exogenous protein.

[0016]

According to some embodiments of the invention, the exogenous protein is selected from the group consisting of a cell marker, a targeting moiety and the pharmaceutical agent.

[0017]

According to some embodiments of the invention, the liposome encapsulates, or attached to a pharmaceutical agent.

[0018]

According to some embodiments of the invention, the pharmaceutical agent is a therapeutic agent.

[0019]

According to some embodiments of the invention, the composition-of-matter is non-immunogenic in a human subject.

[0020]

According to some embodiments of the invention, a cell source of the whole cell membrane fraction comprises cells autologous to a host subject.

[0021]

According to some embodiments of the invention, a cell source of the whole cell membrane fraction comprises cells non-autologous to a host subject.

[0022]

According to some embodiments of the invention, said liposome is attached to a synthetic polymer at an external surface thereof.

[0023]

According to some embodiments of the invention, the pharmaceutical agent is a diagnostic agent.

[0024]

According to some embodiments of the invention, the liposome is unilamellar.

[0025]

According to some embodiments of the invention, the liposome is attached to a synthetic polymer at an external surface thereof.

[0026]

According to some embodiments of the invention, the synthetic polymer is a poly-ethylene-glycol (PEG).

[0027]

According to some embodiments of the invention, the liposome has a size range of 30-1000 nm.

[0028]

According to an aspect of some embodiments of the present invention there is provided a method of producing liposomes comprising,

(a) subjecting cells to hypotonic conditions, so as to obtain ruptured cell membranes and/or ghosts; and

(d) extruding the ruptured membrane and/or ghosts through a matrix of pre-determined porosity.

[0034]

According to some embodiments of the invention, the method further comprises conjugating a synthetic polymer to the liposomes following step (c).

[0035]

According to an aspect of some embodiments of the present invention there is provided a method of encapsulating a pharmaceutical agent in a liposome, the method comprising making the liposomes according to the method above and adding the pharmaceutical agent prior to the step of homogenizing.

[0036]

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the composition-of-matter and a pharmaceutically acceptable carrier.

[0037]

According to an aspect of some embodiments of the present invention there is provided a method of delivering a pharmaceutical agent, the method comprising administering to a subject in need thereof the composition of matter, thereby delivering the pharmaceutical agent.

[0038]

According to some embodiments of the invention, the cell source of the whole cell membrane fraction is autologous to the subject.

[0039]

According to some embodiments of the invention, a cell source of the whole cell membrane fraction is non-autologous to said subject.

[0040]

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying images and drawings. [1-10 images, 11 drawing]. With specific reference now to the images/drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the images/drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

FIGS. 4A-B are Cryo-TEM images of cell-derived liposomes. Cell-derived liposomes were prepared from the cytoplasmatic membranes of hMSCs and were PEGylated by conjugation with monomethoxy-PEG. The resulting PEGylated (FIG. 4A) and un-PEGylated (FIG. 4B) CDLs were then imaged by Cryo-TEM.

FIGS. 7A-B show the binding of CDLs prepared from hMSCs to prostate cancer cell-line (PC3). PC3 cells were labeled with DiO (green) and incubated with CDLs that were previously labeled with DiI (red). Cultures were imaged following 12 hrs incubation. Representative 3D-projection (FIG. 7A) and single-slice (FIG. 7B) images are presented.

[0050]

FIGS. 8A-B are graphs and FACS histograms showing concentration-dependent binding of CDLs prepared from hMSCs to prostate cancer cell-line (PC3). PC3 cells were incubated with various concentrations of CDLs that were previously labeled with a red fluorescent dye (DiI). Following 24 hrs incubation, cells were washed, harvested and analyzed by FACS (FIG. 8A). The mean fluorescence intensity of the cells was calculated and plotted vs. the natural logarithm of CDLs concentration (FIG. 8B).

[0051]

FIG. 9 show the specific binding of CDLs, prepared from conditioned hMSCs (i.e cell cultured with conditioned media of cancer cells), to prostate cancer cell-line (PC3). DiI-labeled CDLs were prepared from hMSCs which were previously incubated for 24 hrs with condition media derived from a prostate cancer cell-line (PC3) and from a non-human cell-line (BHK). The resulting “conditioned” CDLs, as well as CDLs prepared from unconditioned hMSCs (control, NO CM), were incubated with PC3 and BHK cells for 15 min, 1 hr and 3 hrs. Following incubation, the cells were washed, harvested and analyzed by FACS. The percentage in the marker refers to the ratio of DiI-labeled cells within the marker. The percentage in brackets, designated on the upper-left histogram only, refers to the ratio of unlabeled cells within the marker. The marker and the ratio of unlabeled cells within the marker are identical for all histograms.

[0052]

FIGS. 10A-B are cryo-TEM images of hMSCs derived liposomes entrapping soluble Tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). sTRAIL-containing CDLs (FIG. 10A) and empty CDLs (FIG. 10B) were prepared at the same final concentration and imaged by Cryo-TEM under the same conditions. To emphasize CDLs' content, the original grey-scale Cryo-TEM images (left pane) were re-colored to black and white (right pane).

[0053]

FIG. 11 is a schematic illustration of the overall design of targeted carriers based on cell-derived liposomes (CDL). Origin cells that naturally and specifically interact with target cells are selected as a source for cell derived liposomes. For example, MSC membranally interact with cancer cells therefore are selected as a source for cancer targeting carriers. Source cells undergo hypotonic treatment to generate ghost cells, which are then homogenized to produce CDL. Resulting CDL are then able to specifically bind their target cells in a similar manner to the cells they are derived from.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0054]

The present invention, in some embodiments thereof, relates to liposomal compositions and uses of same.

[0055]

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0056]

A major challenge facing cancer therapy is achieving a cytotoxic effect towards cancer cells, while sparing the healthy ones. The importance of the development of novel targeted therapeutic delivery strategies for cancer therapy has long been recognized worldwide.

[0057]

The present inventors have designed a novel delivery vehicle for targeted delivery of therapeutic and diagnostic agents into cells and tissues. The delivery vehicle is liposome-based composed of a whole cell membrane fraction which comprises both natural lipids and proteins. By employing native cell membranes, the delivery vehicles of the present invention may be formulated to be of low immunogenic potential, easily home to the target tissue and can be genetically modified to express therapeutic or targeting moieties.

[0058]

As is illustrated below and in the examples section, which follows and further depicted in FIG. 12, the present inventors have generated liposomes composed of whole cell membranes of mesenchymal stem cells, which are well-known for their homing capacities as well as their immuno-suppressive abilities (i.e., their ability to reduce inflammation and suppress immune cells) and hypo-immunogenic features (i.e., stealth-like features that makes them less immunogenic and less recognizable as foreign matter when heterologously transplanted). The liposomes exhibit the protein signature of mesenchymal stem cells and as such are expected to mediate similar immunosuppression and migratory properties as intact mesenchymal stem cells. These cell derived liposomes were further PEGylated to increase their bioavailability and dispersion and reduce their coagulation. The cell derived liposomes were also treated to encapsulate a therapeutic agent. Altogether these findings, place the present delivery system as a pivotal tool in the diagnosis and treatment of human disease such as cancer.

[0059]

Thus, according to an aspect of the present invention there is provided a composition-of-matter comprising a liposome attached to, or encapsulating a pharmaceutical agent, said liposome being composed of a whole cell membrane fraction.

[0060]

As used herein the term “liposome” refers to fully closed carrier molecules comprising a spherical lipid membrane which itself is in a liquid crystalline phase or a liquid gel phase, in which an entrapped liquid volume is contained. The two liquid phases are immiscible. Thus, liposomes of the present invention (also referred to herein as cell derived liposomes (CDLs), similar to membranes of cells, are in an entirely gel/liquid state and/or liquid crystal state and not in a solid state.

[0061]

The liposomes of some embodiments of the present invention have an expected protein to lipid ration of about 0.8 w/w.

[0062]

Of note, the protein content of hMSCc CDLs is about 0.8 mg/108 cells (as determined by Bradford assay). The lipid content can be easily determined using the Stewart phospholipids assay. It is expected to be about 1 mg/108 cells.

[0063]

The following calcultation can be used to determine the theoretical phospholipids content. Since the dry mass of a single mammalian cell is in the magnitude of 10−7 mg1 and since phospholipids constitutes approximately 10% of the dry cell mass2 then the theoretical yield of the cell derived liposomes' production process (assumed 100% efficiency) should be in the magnitude of 10−8 mg phospholipids per single cell or 1 mg per 108 cells.

According to a specific embodiment of the invention, the liposomes are unilamellar, as determined by Cryo-TEM.

[0067]

According to a specific embodiment of the invention, the liposomes exhibit native membrane symmetry and expression of native markers.

[0068]

Liposomes of the present invention are composed of a whole cell membrane fraction.

[0069]

As used herein the phrase “cell membrane” or “cellular membrane” (which may be interchangeably used) refers to a biological membrane, which surrounds the cell or is an integral part of an organelle thereof (e.g., chloroplast, ER, golgi, mitochondrion, vacuole, nucleus and a lysosome).

[0070]

According to a specific embodiment of the present invention the cell membrane refers to the plasma membrane. The use of plasma membrane is of a specific advantage since it presents proteins, which are associated with cell-to-cell interactions, as well as other recognition molecules, such as receptors that bind soluble ligands.

[0071]

As used herein “a whole cell membrane fraction” refers to a fraction, which does not include lipids alone but also includes membrane proteins.

According to an embodiment of the invention the whole cell membrane fraction also includes carbohydrates.

[0074]

According to a specific embodiment the cell is a eukaryotic cell [e.g., mammalian (such as human), plant, insect cell].

[0075]

According to an additional specific embodiment the eukaryotic cell is a mammalian cell.

[0076]

According to yet an additional embodiment the cell can be a primary cell (i.e., non-immortalized and at times not cultured) or a cell-line.

[0077]

According to yet an additional embodiment the cell can be an embryonic cell.

[0078]

Use of a primary cell may be advantageous for clinical use where non-cultured cells are used in autologous or non-autologous (syngeneic allogeneic or xenogeneic) settings.

[0079]

According to a specific embodiment the eukaryotic cell is a stem cell.

[0080]

As used herein, the phrase “stem cells” refers to cells, which are capable of remaining in an undifferentiated state (e.g., pluripotent or multipotent stem cells) for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (e.g., fully differentiated cells). Preferably, the phrase “stem cells” encompasses embryonic stem cells (ESCs), induced pluripotent stem cells (iPS), adult stem cells, mesenchymal stem cells and hematopoietic stem cells.

[0081]

According to a specific embodiment the stem cell is a mesenchymal stem cell.

[0082]

Mesenchymal stem cells are the formative pluripotent blast cells. Mesenchymal stem cells (MSCs) give rise to one or more mesenchymal tissues (e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts, cardiac like cells) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines. MSCs can be isolated from embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood, bone marrow, adipose and other tissues, although their abundance in the bone marrow far exceeds their abundance in other tissues. MSCs have been shown to have immunosuppressive functions in various settings, including autoimmune diseases and transplantation, rendering liposomes generated therefrom ultimate tools in inflammatory and autoimmune settings.

[0083]

Methods of isolating, purifying and expanding mesenchymal stem cells (MSCs) are known in the arts and include, for example, those disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones E. A. et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

[0084]

Preferably, mesenchymal stem cell cultures are generated by diluting BM aspirates (usually 20 ml) with equal volumes of Hank's balanced salt solution (HBSS; GIBCO Laboratories, Grand Island, N.Y., USA) and layering the diluted cells over about 10 ml of a Ficoll column (Ficoll-Paque; Pharmacia, Piscataway, N.J., USA). Following 30 minutes of centrifugation at 2,500×g, the mononuclear cell layer is removed from the interface and suspended in HBSS. Cells are then centrifuged at 1,500×g for 15 minutes and resuspended in a complete medium (MEM, α medium without deoxyribonucleotides or ribonucleotides; GIBCO); 20% fetal calf serum (FCS) derived from a lot selected for rapid growth of MSCs (Atlanta Biologicals, Norcross, Ga.); 100 units/ml penicillin (GIBCO), 100 μg/ml streptomycin (GIBCO); and 2 mM L-glutamine (GIBCO). Resuspended cells are plated in about 25 ml of medium in a 10 cm culture dish (Corning Glass Works, Corning, N.Y.) and incubated at 37° C. with 5% humidified CO2. Following 24 hours in culture, nonadherent cells are discarded, and the adherent cells are thoroughly washed twice with phosphate buffered saline (PBS). The medium is replaced with a fresh complete medium every 3 or 4 days for about 14 days. Adherent cells are then harvested with 0.25% trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO) for 5 min at 37° C., replated in a 6-cm plate and cultured for another 14 days. Cells are then trypsinized and counted using a cell counting device such as for example, a hemocytometer (Hausser Scientific, Horsham, Pa.). Cultured cells are recovered by centrifugation and resuspended with 5% DMSO and 30% FCS at a concentration of 1 to 2×106 cells per ml. Aliquots of about 1 ml each are slowly frozen and stored in liquid nitrogen.

[0085]

To expand the mesenchymal stem cell fraction, frozen cells are thawed at 37° C., diluted with a complete medium and recovered by centrifugation to remove the DMSO. Cells are resuspended in a complete medium and plated at a concentration of about 5,000 cells/cm2. Following 24 hours in culture, nonadherent cells are removed and the adherent cells are harvested using Trypsin/EDTA, dissociated by passage through a narrowed Pasteur pipette, and preferably replated at a density of about 1.5 to about 3.0 cells/cm2. Under these conditions, MSC cultures can grow for about 50 population doublings and be expanded for about 2000 fold [Colter D C., et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000].

[0086]

MSC cultures utilized by the present invention preferably include three groups of cells, which are defined by their morphological features: small and agranular cells (referred to as RS-1, herein below), small and granular cells (referred to as RS-2, herein below) and large and moderately granular cells (referred to as mature MSCs, herein below). The presence and concentration of such cells in culture can be assayed by identifying a presence or absence of various cell surface markers, by using, for example, immunofluorescence, in situ hybridization, and activity assays.

[0087]

When MSCs are cultured under the culturing conditions of the present invention they exhibit negative staining for the hematopoietic stem cell markers CD34, CD11B, CD43 and CD45. A small fraction of cells (less than 10%) are dimly positive for CD31 and/or CD38 markers. In addition, mature MSCs are dimly positive for the hematopoietic stem cell marker, CD117 (c-Kit), moderately positive for the osteogenic MSCs marker, Stro-1 [Simmons, P. J. & Torok-Storb, B. (1991). Blood 78, 5562] and positive for the thymocytes and peripheral T lymphocytes marker, CD90 (Thy-1). On the other hand, the RS-1 cells are negative for the CD117 and Stro1 markers and are dimly positive for the CD90 marker, and the RS-2 cells are negative for all of these markers.

[0088]

Other cells, which may be used as an effective source for whole cell membrane fraction include, but are not limited to, endothelial cells, hepatic cells, pancreatic cells, bone cells, chondrocytes, neuronal cells and the like.

[0089]

The cells can be used native (i.e., not manipulated by genetic modification) or genetically modified to manipulate the membrane composition of the cell.

[0090]

The advantage of genetic modification is in its increased efficiency. Essentially all (>95%) the CDLs generated from genetically modified cells express the gene-of-interest. The gene-of-interest may be constitutively expressed on the cell source (by integration to the cells genome) or transiently expressed (episomal expression) such as to avoid hazardous implications of stable transfection agents (e.g., lentiviral and retroviral vectors).

[0091]

Thus, the cells may be genetically modified to express a gene-of-interest (i.e., not naturally expressed in the native membrane but also in order to enhance the expression of endogenous proteins that are naturally expressed on the cell's membrane but in lower levels).

[0092]

According to specific embodiments, the gene-of-interest encodes a membrane protein. The gene-of-interest may be a native membrane protein or modified to have a membrane localization signal and other motifs needed for membrane anchorage e.g., a transmembrane domain.

[0093]

Examples of membrane proteins which may be heterologously (exogenously) expressed include, but are not limited to, a targeting protein (e.g., antibodies, receptors, membrane anchored ligands, decoys), a protein which affects the chemistry of the membrane (e.g., structural proteins, charged proteins), a diagnostic protein (e.g., an enzyme as described in length below) and a therapeutic protein (as described in length below).

[0094]

A targeting moiety includes a targeting protein such as an antibody, a receptor ligand and a non-proteinecious molecule such as carbohydrates, which binds cell surface or extra-cellular matrix markers. For example, prostate-specific membrane antigen (PSMA) that is over-expressed on prostate cancer cells can be targeted by its ligand NAAG3 conjugated to a transmembranal motif (e.g, truncated LIME)4. This may be achieved, by genetically engineering the cells (of which the CDLs are derived from) to express the chimeric or natural form of NAAG. For example, the expression plasmid encoding LIME is constructed by PCR and subsequent insertion of the corresponding fragment into pcDNA3.1 (Invitrogen). The primers also have BamHI (5′ primer and 3′ primer) site extension to facilitate the subcloning. The PCR product is digested with BamHI and inserted into corresponding sites in pcDNA3.1(+) (CLONTECH Laboratories, Inc.). For expression vector encoding LIME-acetylaspartylglutamate (NAAG), the open reading frame can be inserted into plasmid coding LIME such that the NAAG is conjugated trough its N-terminus and maintains its C-terminus free to react with PSMA [i.e., LIME(C)—(N)NAAG-COOH] Alternatively, expression plasmid encoding NAAG-LIME chimera can be constructed following the method described previously described for CD8-LIME chimera5. Fragments corresponding to NAAG and LIME transmembrane region were generated by PCR. Primers encoding the 3′ sequences of the NAAG and the 5′ sequences of the LIME fragment were designed to overlap, such that annealing of the two products yielded a hybrid template. From this template, the chimera is amplified using external primers containing XbaI sites. The NAAG-LIME chimera is inserted into pcDNA3.1(+).

[0095]

As used herein, the phrase “surface marker”, refers to any chemical structure, which is specifically displayed at uniquely high density, and/or displayed in a unique configuration by a cell surface or extracellular matrix of the target cell/tissue.

[0096]

For example, the targeting moiety may be useful for targeting to tumor cells. For example, it is generally accepted that the intracellular environment of tumor cells is more alkaline compared to their immediate extracellular environment, which in turn is more acidic than the microenvironment found in the angiogenic blood vessels feeding the tumor. In addition, many previous studies have shown that the surface charges of tumor cells is more negative compared to benign normal cells and even less invasive tumor cells. Accordingly, it may be useful to express membrane-bound enzymes and/or proteins, which will render the liposomes with a positive charge only in the acidic intermediate extracellular environment of the tumor. For example, any membranal protein with a pI of about 7.2-7.4 that falls between the high alkaline pH of the angiogenic blood vessels (pH>7.4) and the low acidic pH of the tumor immediate extracellular environment (pH<7.2) can be used. Such proteins can be specifically identified by cross referencing the RCSB Protein Data Bank (PDB) for human plasma membrane proteins. The expected desirable pI (7.2-7.4) for those proteins can be calculated using the standard iterative algorithm that10, 11 that gives relatively precise results of pI calculations for raw protein sequences12, 13. The algorithm is used in the Compute pI/Mw tool at the ExPASy server. Such liposomes are expected to have negative or neutral charge in the alkaline microenvironment of the angiogenic tumor vessels and positive charge in the more acidic immediate extracellular environment of the tumor. Accordingly, this charge alteration will assist both liposomal extravasation, which is significantly enhanced for negative of neutral particles, and intra-tumor delivery which is more easily accomplished with positively charge particles8, 14, 15.

In a preferred embodiment, the ligand is an antibody or an antibody fragment, targeting antigens specific to a receptor on a target cell. Antibodies can be monoclonal antibodies, polyclonal antibodies or antibody fragments, which are target specific. In an embodiment, the antibodies attached to the liposomes are anti-CD19, anti-CD20, or anti-CD22, for specific binding to a B-cell epitope. These antibodies or antibody fragments are typically derived from hybridomas that show positive reactivity toward the affected B-cells. It is contemplated that other antibodies or antibody fragments targeting any other cell in the body can be similarly used. For example, anti-CD19 antibodies are used to target liposome containing an entrapped agent to malignant B-cells. The antibody recognizes a unique epitope, the CD19 surface antigen, on the B-cells.

[0100]

Methods of expressing heterologous proteins in eukaryotic cells are well known in the art.

[0101]

Thus, an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of the gene-of-interest may be expressed in the cells from which membranes are later extracted. Accordingly, the exogenous polynucleotide sequence may be a DNA or RNA sequence of the gene-of-interest.

[0102]

The phrase “functional portion” as used herein refers to part of the encoded protein (i.e., a polypeptide), which exhibits functional properties of the enzyme such as binding to a substrate. For example, the functional portion of an antibody may be the variable region conferring specificity and additional/or alternatively the constant region, i.e., Fc, which may activate complement and induce cell killing. For example, cells can be transfected with genes encoding one or more members from the GPCRs family (e.g., CCR5, CXCR4 etc.) that will render the liposomes targeted against abundant of cellular pathologies including auto-immune and viral diseases (e.g., HIV/AIDS).

[0103]

To express exogenous gene-of-interest in eukaryotic (e.g., mammalian) cells, a polynucleotide sequence encoding the gene-of-interest is preferably ligated into a nucleic acid construct suitable for eukaryotic cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0104]

Constitutive promoters suitable for use for mammalian expression with the present invention are promoter sequences, which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with the present invention include for example the inducible promoter of the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).

[0105]

The nucleic acid construct (also referred to herein as an “expression vector”) of the present invention includes additional sequences, which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.

[0106]

The nucleic acid construct of the present invention typically includes a signal sequence for directing the translated polypeptide to the membrane and additionally a membrane anchor domain such as a transmembrane domain or a lipid based anchor (e.g., GPI).

[0107]

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

[0110]

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0111]

Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.

[0112]

In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

[0113]

The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

[0114]

The expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0115]

Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0116]

Expression vectors containing regulatory elements from eukaryotic viruses such as lentiviruses and retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0117]

Alternatively, cells, membranes, ghosts or CDLs (either of which may be native or genetically modified), may be chemically treated such as to present a protein, a saccharide, a synthetic polymer, a peptide or any combination of same. Methods of modifying the membrane with a synthetic polymer are described herein below and in the examples section, which follows. Such a chemical attachment may be effected at any stage from live cultured or suspended cells to produced CDLs.

[0118]

For example, the CDLs may be also chemically conjugated with folate that may further enhance their targeting and attachment to tumor cells, which are known to express higher levels of folate receptors compared to benign cells.

[0119]

According to another example, it is also possible to permanently modulate the CDLs to have a more positive surface charge by treating them with cations, salts or polycations (e.g., Polybrene®, polyethyleneimine and Poly-L-Lysine) rendering them more positive to better target the tumor angiogenic vasculature.

[0120]

Non-native material can be also introduced to the surface of the CDLs by fusion (e.g., PEG or detergent induced) with other liposomes (e.g., cell-derived or synthetic) that may be comprised of well characterized lipids, proteins and additives. Such fusion, creating hybrid CDLs, can be used to conjugate any moieties (e.g., targeting, therapeutic, diagnostic, stealth-rendering etc.) to the CDLs and to alter their surface properties. See Example 5 for further guidance on liposomal fusion.

[0121]

Synthetic polymers are typically used to prevent or reduce coagulation, increase dispersion, reduce interaction with blood components, evade non-specific uptake by the mononuclear phagocytic system and prolong the particle circulation time to a large extent thus, rendering the liposomes with properties and features that are commonly referred to as stealth properties or long-circulating liposomes. Accordingly, the pH nano-environment at the particle surface may also be dependent upon the length of these molecules.

The polymers may be employed as homopolymers or as block or random copolymers.

[0124]

The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually distearylphosphatidylethanolamine (DSPE).

[0125]

A specific family of lipopolymers, which may be employed by the invention include PEG-DSPE (with different lengths of PEG chains) in which the PEG polymer is linked to the lipid via a carbamate linkage and Polyethyleneglycol distearoylglycerol. The PEG moiety headgroup preferably has a molecular weight from about 750 Da to about 20,000 Da. More preferably, the molecular weight is from about 750 Da to about 12,000 Da and most preferably between about 1,000 Da to about 5,000 Da. Two exemplary DSPE-PEG are those wherein PEG has a molecular weight of 2000 Da, and of 5000a designated herein DSPE-PEG(2000) (DSPE-PEG2k) and DSPE-PEG(5000) (DSPE-PEG5k).

[0126]

Specific families of lipopolymers, which may be also employed by the invention, include C8 and C16 mPEG Ceramides (with different lengths of PEG chains) in which the PEG-Ceramides contain ester linkages between the PEG and ceramide moieties that allow the compound to be easily metabolized. The PEG moiety headgroup preferably has a molecular weight from about 750 Da to about 2,000 Da. More preferably, the molecular weight is about 2,000 Da.

[0127]

Conventional post-insertion PEGylation of common liposomes requires heating or solublization in a detergent containing solution that might damage surface proteins and lead to encapsulate leakage. Therefore, CDLs may be also PEGylated by the two following described methods or their combination. Primarily, PEGylated CDLs will be prepared by detergent-dialysis incorporation of PEGylated lipids into the ghost cell membrane (prior to CDLs preparation). Following, direct PEGylation of the CDLs may be performed with monomethoxy-PEG activated by succinimidyl succinate, which has been proven to increase the trasnfection efficiency and reduce serum mediated inactivation of PEGylated lentiviral particles, used as gene transduction vectors16.

[0128]

Chemical binding of non-proteinaceous components (e.g., synthetic polymers, carbohydrates and the like) to the liposomal surface may be employed. Thus, a non-proteinaceous moiety, may be covalently or non-covalently linked to, embedded or adsorbed onto the liposome using any linking or binding method and/or any suitable chemical linker known in the art. The exact type and chemical nature of such cross-linkers and cross linking methods is preferably adapted to the type of affinity group used and the nature of the liposome. Methods for binding or adsorbing or linking the enzyme and/or targeting moiety are also well known in the art.

[0129]

For example, the enzyme and/or targeting moiety may be attached to a group at the interface via, but not limited to, polar groups such as amino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides. In addition, the enzyme and/or targeting moiety may be attached via, but not limited to, active groups such as succinimidyl succinate, cyanuric chloride, tosyl activated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol, etc. Moreover, the enzyme and/or targeting moiety may be attached via, but not limited to, hydrophobic bonds (Van Der Waals) or electrostatic interactions that may or may not include cross-linking agents (e.g., bivalent anions, poly-anions, poly-cations etc.).

[0130]

Once the cell source is available the liposomes are made. Thus, there is provided a method of producing liposomes comprising,

The method may be practiced according to other well accepted protocols known in the art such as that of Boone, C. W., Ford, L. E., Bond, H. E., Stuart, D. C. & Lorenz, D. Isolation of plasma membrane fragments from HeLa cells. J Cell Biol 41, 378-392 (1969); and Westerman and Jensen Methods Enzymol. 2003; 373:118-27 (each of which is incorporated herein by reference) with or without modifications.

[0134]

As used herein, the term “ghosts” refers to a cell that all of its cytoplasmic contents and/or nucleolus were removed by cell lysis and/or membrane rapture so that only its outer cytoplasmatic/cell membrane remains; and

[0135]

Without being bound to a specific protocol it is suggested in a specific embodiment that liposomes of the present invention are made in a step-wise manner. First, plasma membranes are isolated from cells (109 cells) primarily by using hypotonic treatment such that the cell ruptures and ghost cells are formed. Altrnatively, ghost cells can be formed using mild sonication, freeze-thaw, French-press, needle-passaging or solublization in detergent-containing solutions. According to a specific embodiment hypotonic treatment is effected in Tris-magnesium buffer (e.g., pH 7.4 or pH 8.6 at 4° C., pH adjustment made with HCl). Cell swelling is monitored by phase-contrast microscopy. Once the cells swell and ghosts are formed, the suspension is placed in a homogenizer. Typically, about 95% cell rupture is sufficient. The membranes/ghosts are then placed in Sucrose (0.25 M or higher) for preservation. To avoid adherence, the ghosts are placed in plastic tubes and centrifuged. A laminated pellet is produced in which the topmost lighter gray lamina consists only entirely of ghosts. However, the entire pellet is processed, to increase yields. Centrifugation (e.g., 3,000 rpm for 15 min at 4° C.) and washing (e.g., 20 volumes of Tris magnesium/TM-sucrose pH 7.4) may be repeated.

[0136]

In the next step, the ghost fraction is separated by floatation in a discontinuous sucrose density gradient. A small excess of supernatant is left over the washed pellet, which now contains ghosts, nuclei, and incompletely ruptured whole cells. Additional 60% w/w sucrose in TM, pH 8.6 is added to the suspension to give a reading of 45% sucrose on a refractometer. After this step, all solutions contain TM pH 8.6. 15 ml of suspension are placed in SW-25.2 cellulose nitrate tubes and discontinuous gradient is formed over the suspension by adding 15 ml layers, respectively, of 40% and 35% w/w sucrose, and then adding 5 ml of TM-sucrose (0.25 M). The material is now centrifuged at 20,000 rpm for 10 min, 4° C. The nuclei sediment form a pellet, the incompletely ruptured whole cells are collect at the 40%-45% interface, and the ghosts are collected at the 35%-40% interface. The ghosts are collected and pooled.

[0137]

In the next step, the ghosts are homogenized such as by sonication which may be followed by extrusion.

[0138]

A specific sonication protocol relates to 5 second sonication using an MSE sonicator with microprobe at an amplitude setting of 8 (Instrumentation Associates, N.Y.). This short period of sonication is enough to cause the plasma membrane of the ghosts to break up into cell derived liposomes (CDLs). Under these specific conditions organelle membranes are not disrupted and these are removed by centrifugation (3,000 rpm, 15 min 4° C.). Plasma membrane vesicles (CDLs) are then purified by sedimentation in a continuous sucrose density gradient.

[0139]

Liposomes comprising one or more pharmaceutical agent of the present invention are preferably in the size range of 20-1000 nm e.g., 30-1000 nm, 0.02-1.0 μm, more preferably 0.05-1.0 μm, more preferably 0.07-0.5 μm and more preferably 0.1-0.3 μm. An advantage of liposomes smaller or about 0.2 μm is that they can easily permeate through tumor vasculature (due to the EPR effect), they are not readily uptaken by macrophages and they can undergo filter sterilization.

[0140]

Extrusion of liposomes through a commercially available polycarbonate membrane (e.g., from Sterlitech, Washington) or an asymmetric ceramic membrane (e.g., Membralox), commercially available from Pall Execia, France is an effective method for reducing liposome sizes to a relatively well defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes (e.g., 400 nm, 100 nm and/or 50 nm pore size) to achieve a gradual reduction in liposome size and uniform distribution.

[0141]

At any step prior to the homogenization, sonication and/or extrusion, that is, typically following ghosts preparation, a pharmaceutical agent may be added to the reaction mixture such that the resultant liposomes encapsulate the pharmaceutical agent.

[0142]

As used herein the phrase “pharmaceutical agent” refers to a therapeutic agent or diagnostic agent, which can be used to treat or diagnose a medical condition, respectively.

[0143]

According to a specific embodiment, the composition comprising the pharmaceutical agent and the liposome is hypo or non-immunogenic especially when the cell source is a mesenchymal stem cell.

[0144]

Thus, the liposome of the present invention may have a pharmaceutical agent adsorbed to a surface thereof or encapsulated therein either within the intra-liposomal polar phase or the lamellar non-polar lipid phase.

[0145]

Methods of conjugating molecules (e.g., targeting moieties, pharmaceutical agents, synthetic polymers and the like) to liposomes are well known in the art. For example, a the pharmaceutical agent (or any other molecule) may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from based on hydrophobic interactions (Van Der Waals bonds) or electrostatic interactions with or without the use of cross-linking agents (e.g. anions and poly-anions). Hydrophobic and/or amphipathic pharmaceutical agent (or any other hydrophobic and/or amphipathic molecule) may be soulibilized, partially soulibilized or partitioned into the cells, ghosts or liposomal lipid membranes with or without the use of detergent and/or by detergent dialysis. A pharmaceutical agent (or any other molecule) may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from based on covalent bonds with active groups. A pharmaceutical agent may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from as a conjugate of an antibody or part of that specifically recognized a natural moiety found on the liposomes, ghosts or cells. For example, pharmaceutical agent may be adsorbed to the surface (inner or outer) of the liposomes via, but not limited to, polar groups such as amino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides. In addition, the pharmaceutical agents may be adsorbed via, but not limited to, active groups such as succinimidyl succinate, cyanuric chloride, tosyl activated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol, etc.

[0146]

Entrapped in, adsorbed, expressed, conjugated, attached, and/or solubilzed on the liposomes' surface or membrane is a therapeutic agent for delivery to the target cells and/or tissues by one or more of, but not limited to, the following mechanisms:

[0147]

Direct intracellular delivery of the agent by means of membrane fusion between the liposomes and cells and/or liposomal uptake by endocytosis, phagocytosis or by any kind of transmembranal transport mechanism.

[0148]

Diffusion and/or leakage of the agent from the liposome and consequent binding to the surface of the target cells/tissue and/or uptake into the target cell/tissue by diffusion, endocytosis, phagocytosis or by any kind of transmembranal transport mechanism.

[0149]

Binding to the surface of the target cells and/or tissues of an agent which is permanently, constantly or transiently expressed, attached, adsorbed, conjugated and/or solubilzed on the liposomes' surface or membrane.

[0150]

A variety of therapeutic agents can be entrapped in lipid vesicles, including water-soluble agents that can be stably encapsulated in the aqueous compartment of the liposome, lipophilic compounds that stably partition in the lipid phase of the vesicles, or agents that can be stably or transiently attached, conjugated, adsorbed or expressed on to the outer or inner surfaces of the liposomes, e.g., by electrostatic, covalent or hydrophobic interactions.

As mentioned above, the therapeutic agent may be a protein, such as an enzyme which compensates for loss in activity or poor expression of an endogenous enzyme e.g., the enzyme hexosaminidase A, a shortage of which results in Tay-Sachs disease.

The liposome-entrapped compound may also be a diagnostic agent such as an imaging or a contrast agent as indium and technetium, enzymes such as horseradish peroxidase and alkaline phosphatase, MRI contrast media containing gadolinium, X-ray contrast media containing iodine, ultrasonography contrast media such as CO2, europium derivatives, fluorescent substances such as carboxyfluorescein and illuminants such as N-methylacrydium derivatives.

[0155]

Once the liposomes are formed (i.e., with or without a pharmaceutical agent), they may be characterized for their size distribution, composition, concentration, zeta potential, electrical surface potential, surface (local) pH, protein to lipid ratio and therapeutic efficacy in vitro and in vivo.

[0156]

Experimentally tested liposomes of the present invention have the following size values as described on Table 2 below:

[0000]

TABLE 2

Without PEGylation:

With PEGylation:

Avg. by number 30 nm

Avg. by number 100 nm

Avg. by volume 200 nm

Avg. by volume 215 nm

Aggregation factor: 200/30 = 7

Aggregation factor: 215/100 = 2

[0157]

Empty liposomes or liposomes comprising one or more pharmaceutical agent of the present invention are preferably in the size range of 30-3000-nm, more preferably 50-500 nm, more preferably 30-300 nm, more preferably 50-200 nm and more preferably 70-150 nm. An advantage of liposomes smaller or about 100-nm is its ability to penetrate through very narrow blood vessels which is of great significance in diagnostic and treatment.

[0158]

Any method known in the art can be used to determine the size of the liposome. For example, a Nicomp Submicron Particle Sizer (model 370, Nicomp, Santa Barabara, Calif.) utilizing laser light scattering can be used. Other methods of measuring liposome size include photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS), light obscuration methods (Coulter method, for example), rheology, or microscopy (light or electron). The preferred average effective particle size depends on factors such as the intended route of administration, formulation, solubility, toxicity and bioavailability of the compound.

Thus, liposomes of the present invention are characterized by a zeta potential of −20 to −15 mV without PEGylation and −15 to −10 mV with PEGylation.

[0162]

As mentioned, liposomes of the present invention are advantageously used in the clinic.

[0163]

Thus, according to an aspect of the invention there is provided a method of delivering a pharmaceutical agent, the method comprising administering to a subject in need thereof the above-describe liposome, wherein the pharmaceutical agent is enclosed therein or adsorbed thereon, thereby delivering the pharmaceutical agent.

[0164]

According to an embodiment, the cells are target cells and the liposomes contain a targeting moiety, either chemically conjugated, heterologously added, as described above, or natively presented in the membranes from which the liposome is comprised (e.g., as in MSCs, which migrate to tumor cells).

[0165]

The cell source for the liposomes may be autologous or non-autologous (e.g., allogeneic, xenogeneic) to the subject.

[0166]

The “target cell” referred to herein is a cell or a cluster of cells (of homogenous or heterogeneous population) and/or tissue to which a substance is to be delivered by using the liposome. Examples thereof include cancer cells, vascular endothelial cells of angiogenic cancer tissues, cancer stem cells, interstitial cells of cancer tissues, cells affected by genetic abnormality, cells infected by a pathogen and the like. The “target molecule” may be any molecule presented the surface of the target cells or cells adjacent to the target cells. Another form of the target molecule includes molecules which are released from cells. Examples thereof includes extracellular matrix components, secretions or architectures of cancer cells or interstitial cells of cancer tissues, and specific examples thereof include tumor markers, structures between cells and the like.

[0167]

Delivering can be for diagnostic reasons (e.g., the liposome includes a diagnostic agent) or for treating (i.e., as a drug delivery tool, delivering a therapeutic agent).

[0168]

The liposomes may be administered to the subject per se, or as part of a pharmaceutical composition.

[0169]

As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the active ingredients to the subject.

[0170]

Herein the term “active ingredient” refers to the therapeutic agent (with or without the liposome) accountable for the biological effect. It is to be appreciated that the liposome per se may have immunomodulatory function such as when prepared from membranes of MSCs or other immunomodulatory cells (e.g., immune B and T lymphocytes etc.). It is also to be appreciated that the liposome per se may have a cytoxoic effect on the target cells as due to membrane fusion with target cells and consequent disruption to cell membrane, cytoskeleton and functions. In such a case measures are taken to include a targeting moiety such that the cytotoxic effect becomes specific.

[0171]

Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered active ingredients. An adjuvant is included under these phrases.

[0172]

Herein, the term “excipient” refers to an inert substance added to the pharmaceutical composition to further facilitate administration of an active ingredient of the present invention or to increase shelf-life stability. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and salts and types of starch, cellulose derivatives, gelatin, vegetable oils, EDTA, EGTA, Poly-L-Lysine, polyethyleneimine, Polybrene (hexadimethrine bromide), polyethylene glycols and other poly or single anions. The pharmaceutical composition may advantageously take the form of foam, aerosol or a gel.

[0173]

Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0174]

Suitable routes of administration include any of various suitable systemic and/or local routes of administration.

The pharmaceutical composition may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0177]

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0178]

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0179]

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0180]

For oral administration, the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0181]

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.

[0182]

Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0183]

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0184]

For administration via the inhalation route, the active ingredients for use according to the present invention can be delivered in the form of an aerosol/spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., a fluorochlorohydrocarbon such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane; carbon dioxide; or a volatile hydrocarbon such as butane, propane, isobutane, or mixtures thereof. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the active ingredients and a suitable powder base such as lactose or starch.

[0185]

The pharmaceutical composition may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0186]

A pharmaceutical composition for parenteral administration may include an aqueous solution of the active ingredients in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0187]

Alternatively, the active ingredients may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0188]

The pharmaceutical composition may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0189]

The pharmaceutical composition should contain the active ingredients in an amount effective to achieve disease treatment.

[0190]

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0191]

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture and in vivo assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0192]

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

[0193]

Dosage amount and interval may be adjusted individually to provide plasma or brain levels of the active ingredients which are sufficient to achieve the desired therapeutic effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0194]

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0195]

The amount of the composition to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0196]

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredients. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

[0197]

As used herein the term “about” refers to ±10%.

[0198]

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0199]

The term “consisting of” means “including and limited to”.

[0200]

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0201]

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0202]

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0203]

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0204]

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0205]

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0206]

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0207]

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

[0208]

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Human MSCs were purchased from Lonza® (Switzerland) and characterized using Giemsa staining and FACS analysis for human mesenchymal stem cells (hMSCs) typical cell surface markers. As seen from FIGS. 1A-C the cells appear to be positive for CD90, CD105, CD44, and CD29 and negative for CD133, CD31, CD34 and CD144, as expected for hMSCs.

[0211]

The migratory abilities of the hMSCs towards cancer cells were tested as well. For these experiments, hMSCs were labeled by a red-fluorescent dye (DiI) while several other cell lines (including a prostate cancer cell-line—PC3) were labeled by a green fluorescent dye (DiO). Labeled cells were drop-wise seeded on tissue culture plates, incubated for 72 hrs and imaged using the Maestro in vivo Imager (FIG. 2). As seen, specific migration of hMSCs towards PC3 cancer cells was demonstrated while “avoiding” interaction with other cell-lines (BHK, Cf2Th, and COS-7).

[0212]

Additional experiments were conducted to validate the targeting abilities of conditioned hMSCs to breast cancer cell-line MCF7. For that, human hMSCs were cultured with or without MCF7-derived conditioning media, labeled with Dil (red) and co-cultured with MCF7 cells labeled with DiO (green). Following 2 hr incubation, cultures were washed and the coverage areas of each cell type and cell overlay (yellow) were determined using fluorescent microscopy image analysis. Assuming that the overlay of Dil and DiO is a consequence of physical interaction between the two cell types, the percent of overlay may represent the amount of membranal interactions between the two cell types. As can be seen from FIG. 3, the incubation of conditioned hMSCs with MCF7 cells resulted in 7% overlay, out of the total cell coverage area, compared to no overlay when using unconditioned hMSCs (p<0.001). This apparent targeting evidently differs from the migration described in FIG. 2 as it is mainly governed by membranal interactions between the hMSCs and the cancerous cells and not based on the migratory abilities of hMSCs that is largely mediated by soluble factors.

Cell Derived Liposome preparation—About 107 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl2) pH 7.4 for 15 min at 4° C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy). For stabilizing the ghosts' suspension, 60% (w/w) sucrose solution was immediately added to the suspension to make a final concentration of 0.25M or 10% by volume. Ghosts were then centrifuged at 3000 rpm for 15 min at 4° C. The supernatant was discarded and the pellet of ghosts was then washed twice with 0.25 M sucrose in TM-buffer pH 7.4, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. In order to create sonicated ghosts, the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 min at 4° C. The pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. For the formation of unilamellar liposomes, the resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 μm and 0.1 μm (Osmonics Inc., Minnesota USA). The extruded liposomes were then centrifuged for 45 min at 150,000 g at 4° C. The supernatant was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.

[0214]

Cell derived liposomes surface protein PEGylation (according to the methof of Croyle, M. A. et al., 2004)—About 107 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl2) pH 7.4 for 15 min at 4° C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy). For stabilizing the ghosts' suspension, 60% (w/w) sucrose solution was immediately added to the suspension to make a final concentration of 0.25M or 10% by volume. Ghosts were then centrifuged at 3000 rpm for 15 min at 4° C. The supernatant was discarded and the pellet of ghosts was then washed twice with 0.25 M sucrose in TM-buffer pH 7.4, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. In order to create sonicated ghosts, the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 min at 4° C. The pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. For the formation of unilamellar liposomes, the resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 μm and 0.1 μm (Osmonics Inc., Minnesota USA). The extruded liposomes were then centrifuged for 45 min at 150,000 g at 4° C. The supernatant was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.

[0215]

The protein content on the liposome's surface was determined using the Bradford protein assay, referring to bovine serum albumin (BSA) as standard. Succinimidyl succinate activated Monomethoxy-PEG was obtained from Sigma Chemicals (St. Louis, Mo.) and was added to the resuspended liposomes at a 10:1 ratio relative to the liposomes' protein content as previously determined by the Bradford assay. For example, 10 μg of Monomethoxy-PEG were added for each 1 μg of protein. The conjugation reaction between the Monomethoxy-PEG and the liposomes was performed at 25° C. with gentle agitation. The reaction was stopped by the addition of 10× L-lysine (Sigma Chemicals) with respect to the amount of Monomethoxy-PEG added. Un-reacted free PEG, excess lysine, and reaction byproducts were eliminated by buffer exchange over a Micro-Bio Spin P-30 chromatography column (Bio-Rad) equilibrated with TM buffer pH 8.6.

Method: Liposomes were created form 2×107 hMSCs as previously described. Tosyl-activated paramagnetic Dynabeads® M-280 (invitrogen) were used as they were able to non-specifically and covalently bind any protein and/or liposomes conjugated with proteins and to be later analyzed by flow-cytometry. Using magnetic separation device (MACS, Dynal™ Magnetic Particle Separator—Invitrogen), the beads were washed with the coupling buffer. To increase their ability to conjugate proteins, the beads were then further washed with 3M ammonium sulfate added to the coupling Buffer. later, 4 samples were prepared containing 107 beads each: Beads only, beads with liposomes, beads with liposomes to be labeled with secondary antibody (isotype control) and beads conjugated with liposomes to be labeled with primary and secondary antibody (test sample). About 5×106 cell equivalent liposomes were added to each sample. Liposomes and beads were then incubated for at least 12 hr at 4° C. After attachment, samples were re-suspended in the washing\blocking buffer. Each sample was suspended in total volume of 200 μl. First, mouse MABs anti-human CD29, CD44, CD90 or CD105 were added to the appropriate samples in a ratio 1:100. Samples were incubated for 30 min in RT. Next, samples were washed twice using the magnetic device. Then secondary ABs (FITC-conjugated goat anti mouse) were added and the samples were incubated for 30 min at RT in the dark. All antibodies, primary and secondary, were purchased from BD—Becton, Dickinson and Company. Following washing of the samples as mentioned before, the samples were run and analyzed using FACSCalibur and CellQuest Pro (BD).

[0218]

Results

[0219]

PEGylated Cell-Derived Liposomes (PEG-CDLs) are expected to be protected from opsonization and degradation, thus, having stealth properties and longer circulation time in vivo. Also, PEGylation may reduce the risk of non-specific binding and fusion of liposomes as with non-target cells17-19.

[0220]

Cryo-TEM imaging of the CDLs demonstrated that the PEGylation had no apparent effect on the desirable small unilamellar morphology of the CDLs (FIGS. 4A-B). However, the PEGylated liposomes (FIG. 4A) seemed more dispersed and less coagulated than the un-PEGylated liposomes (FIG. 4B), that were imaged at the same concentration and under the same conditions. Apparently, not only that the PEGylation does not damage liposomes' morphology but it may also improve their dispersion and stability. The size and size distribution of the CDLs were further analyzed using number and volume weighing DLS analysis (Dynamic Light Scattering, Malvern Nanosize). While number-weight DLS analysis (FIG. 5A) demonstrated an increase in liposomes' size following PEGylation (from ˜30 nm to ˜100 nm), volume-weight DLS analysis (FIG. 5b) demonstrated that the addition of PEG had a homogenizing effect on the system, exhibiting a significant reduction in the liposomes' size distribution. Evidently, the addition of PEG groups stabilized the system and prevented aggregation even though the Zeta-potential decreased from −17.9 mV to −10.2 mV (FIG. 5C).

[0221]

Finally, the expression of MSC-specific surface markers, on the surface of hMSCs derived liposomes, was validated by FACS analysis (FIG. 6). As seen, the CDLs retained their cytoplasmatic membrane symmetry and the expression of correctly oriented typical hMSCs surface markers (i.e., CD44, CD29, CD90 and CD105).

Example 3Binding and Specific Targeting of Cancerous Cell-Lines by CDLs

[0222]

Confocal microscopy imaging and flow cytometry analysis were used to determine the binding of fluorescently labeled CDLs prepared from hMSCs to prostate cancer cells (PC3). As can be seen from FIG. 7A, most vesicles favored cell binding. In addition, the vesicles were detected inside and fused with the cell membranes (FIG. 7A).

[0223]

Flow cytometry analysis demonstrates that most cells bind the vesicles (FIG. 8A) in a concentration-dependent manner (FIG. 8A), thus allowing to determine the extent of liposomal binding according to the cells' mean fluorescence intensity.

[0224]

To test the specific targeting of cancerous cell-lines, DiI-labeled CDLs were prepared from hMSCs, which were previously incubated for 24 hrs with condition media derived from a prostate cancer cell-line (PC3) and from a non-human cell-line (BHK). The resulting “conditioned” CDLs, as well as CDLs prepared from unconditioned hMSCs (control), were incubated with PC3 and BHK cells for 15 min, 1 hr and 3 hrs. Following incubation, cells were washed, harvested and analyzed by flow cytometry (FIG. 9).

[0225]

The specificity index for every experiment, given a certain conditioning media

[0226]

(NO CM, BHK-derived and PC3-derived) and incubation time (15 min, 1 hr and 3 hrs), was calculated according to the following equation:

The specificity index results, summarized in Table 3 below, not only illustrates that the system exhibits specificity towards cancerous cells but that this specificity, as excepted, decreases with incubation time. In addition, the specificity index values show that the system's specific affinity towards cancer cells can be largely affected by subjecting the cells to various conditioning media prior to CDLs preparation.

Equipment—Amicon Ultra-15 centrifugal filters (Millipore number UFC901024); and French Press cell disruption system. All solutions were filtered for sterility through a 0.2 μm filters; all procedures up to Day 2 (step 4, pellet of bacteria after IPTG O/N induction) were carried out in a Sterile Hood.

STEP 2: The “starter” culture was spun down at 1000 g for 15 min to remove the antibiotics. The supernatant was discarded and resuspended in 40 ml of fresh 2YT. In a Sterile Hood, the resuspended 40 ml of the O/N preparation from step number 1 was added to 2 L of 2YT in a 4 L flask (alternatively add the resuspended pellet of 20 ml of the O/N preparation from step number 1 to two 2 L flasks each containing 1 L of 2YT). The solution was incubated for 2-3 hours in a 37° C. shaking incubator at 250 RPM. Measures are taken not incubate for more than 3 hours until O.D. is 2.5-3.0 (it is recommended to measure O.D.595 after 2 hours).

[0236]

STEP 3: Just before IPTG induction, PBS was added to a final concentration of 0.1× to maintain the pH of the culture. EtOH (99% Dehydrated) was added to a final concentration of 2% (40 ml in 2 L culture) to increase the solubility of the protein. 10 ml/L of 0.5M Glucose was added as a carbon source to a final concentration of 5 mM.

[0237]

STEP 4: 500 μM IPTG were added to the supplemented culture. The culture was incubated over night in a shaking incubator (250 RPM) at 20-25° C.

STEP 6: Cells were lyzed by running the bacteria from step number 5 twice through a French Press cell disruption system. Alternatively, 10 ml aliquots in 50 ml tubes were sonicated on ice at 30% power by 4 bursts of 10 sec each. After Cell disruption, the following was added to each 40 ml of cell lysate: 0.1% Triton-X (40 μl of TritonX100), 1 mM MgCl2 (40 μl of 1M stock MgCl2) and 1 mM DTT (40 μl of 1M stock DTT). The solution was mixed thoroughly and incubated at RT for 15 min on a rocker or shaker.

[0241]

STEP 7: The bacterial lysate was spun down for 10 min at 16,900 g and 4° C. Supernatants were aspirated and collected in 50 ml tubes.

[0242]

STEP 8: Binding to GSH (Glutathione—Sepharose 4B Beads)—In a 15 ml Falcon Tube, 3 ml of GSH Beads were washed three times with PBS. Collected supernatant was centrifuged again because of mass bead loss. The washed beads were added to the bacterial cell lysate from step number 7 and incubated with tumbling for 1 hour at 4° C.

[0243]

STEP 9: The bacterial cell lysate, containing the sepharose beads from step number 8, was spun down at 2000 RPM for 1 min in a MULTI CENTRIFUGE CM 6M ELMI to separate the protein-conjugated beads from the cell-lysate. The supernatant was collected and was centrifuged again to pellet the remaining sepharose beads in the supernatant (that might have not pelleted during the first centrifugation). The pellet from both centrifugations, containing the sTRAIL-conjugated beads, was washed 5 times with 5 ml of PBS supplemented with 0.1% Triton-X100, 150 mM NaCl and 1 proteinase inhibitor tablets per 20 ml PBS.

[0244]

STEP 10: Elution of GST-sTRAIL—The beads were spun down as before and the supernatant was aspirated. 3 ml of 50 mM Glutathione (pH 8.5) in 10 mM Tris-HCl and 100 mM NaCl were added. Each 3 ml was vortexed for 2 min and the protein was eluted into the supernatant. The supernatant was aspirated as before and the supernatant kept. The procedure of elution was repeated 3-4 times.

[0245]

STEP 11: The protein was concentrated using Amicon Ultra-15 10K NMWLnumber UFC9010, giving a protein yield of about 5 mg/L culture. sTRAIL was produced at a final concentration of 0.2 mg/ml

[0246]

sTRAIL entrapment—About 107 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl2) pH 7.4 for 15 min at 4° C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy). For stabilizing the ghosts' suspension, 60% (w/w) sucrose solution was immediately added to the suspension to make a final concentration of 0.25M or 10% by volume. Ghosts were then centrifuged at 3000 rpm for 15 min at 4° C. The supernatant was discarded and the pellet of ghosts was then washed twice with 0.25 M sucrose in TM-buffer pH 7.4, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. In order to create sonicated ghosts, the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 min at 4° C. The pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4° C. After, sTRAIL was added to the suspended sonicated ghosts (in TM buffer pH 8.6) to a final concentration of 1 μg per 1 ml of ghost suspension. For the formation of unilamellar liposomes containing sTRAIL, the sTRAIL-containing resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 μm and 0.1 μm (Osmonics Inc., Minnesota USA). The extruded liposomes containing sTRAIL were then centrifuged for 45 min at 150,000 g at 4° C. The supernatant containing excess non-encapsulated sTRAIL was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.

[0247]

Results

[0248]

TRAIL—tumor necrosis factor-related apoptosis-inducing agent is a type II transmembrane protein that induces apoptosis in tumor cells of diverse origins, while sparing most normal cells20-24. Delivery of both full length and truncated, secreted forms of TRAIL (sTRAIL) were shown to induce apoptosis in a variety of cancer cells both in culture and in vivo25, 26. Our preliminary experiments with sTRAIL included its production and passive encapsulation within hMSCs CDLs at a final concentration of 1 μg/ml. Cryo-TEM imaging of the resulting sTRAIL-containing CDLs (FIG. 10A left pane), compared to empty CDLs (FIG. 10B, left pane) prepared and imaged under the same conditions, demonstrates the accumulation of 14-20 nm protein micelles within the CDLs. The sTRAIL micelles are even more clearly visible after digitally re-coloring the images from grey-scale to black-and-white (FIGS. 10A and 10B, right panel).

Example 5Preparation of “Hybrid CDLs” by Fusion with Other Liposomes

[0249]

Various molecules (e.g., proteins, lipids, additives and even encapsulates) can be introduced, conjugated or attached onto the surface of the CDLs by means of fusion between the CDLs and other liposomes (synthetic or cell-derive), thus creating—“Hybrid CDLs”. For example, a liposomal formulation made from synthetic well characterized lipids may be conjugated with a protein on its surface or may contain a desirable encapsulate. Then, by means of induced membrane fusion between the said synthetic liposomes and CDLs a hybrid CDL may be formed. These hybrid CDLs contain both lipids and proteins from the cell-membrane they derive from and the lipids and proteins that were originally formulated on the fused synthetic liposomes. Such introduction of ‘non-native’ materials onto the Hybrid CDLs may be used to attach or conjugate any molecule or moieties related, but not limited to, liposomal targeting, therapeutic effect, diagnostic effect, stealth-rendering properties etc. Such fusion may be also used to change the biochemical or chemophysical properties of the CDLs membranes by introduction of synthetic lipids, additives (e.g., cholesterol, ceramides) etc. Such fusion may be also used to increase the encapsulation efficiency in the said CDLs. Since encapsulation in CDLs is mainly limited to passive encapsulation, fusion with synthetic liposomes that were actively loaded with high concentration of encapsulates may significantly improve the CDLs' encapsulation efficiency.

[0250]

Methods for preparation of synthetic liposomes are well known in the art and mainly include hydration of dehydrated lipids to form lamellar structures and consequent homogenization of those lamellar structures to create liposomes. Synthetic liposomes of the said application can be produced by any method known in the art including, but not limited to, solvent evaporation, solvent replacement, detergent dialysis, extrusion, sonication, freeze-drying, reverse phase evaporation, ethanol/ether injection, agitation and/or any other form of mechanical homogenization. Liposomes can be prepared from a variety of synthetic and naturally derived lipids and may or may not contain additional additives (e.g., cholesterol, ceramides etc.). Methods for active encapsulation of matter in such synthetic liposomes, which are mainly based on membrane pH gradient or active transporters, are also well known in the art and may be used to create synthetic liposomes with high encapsulation efficiency.

[0251]

Fusion between CDLs and other liposomes to create “Hybrid CDLs” can be readily and easily accomplished by adding short chain free PEG (˜200-500 Da) to the liposomes. The mechanism of PEG-induced vesicle fusion is believed to be related to the reduction of water activity and the dehydration of the lipid headgroups which consequently leads to vesicle coagulation and fusion. Fusion can also be artificially induced through electroporation in a process known as electrofusion. It is believed that this phenomenon results from the energetically active edges formed during electroporation, which can act as the local defect point to nucleate stalk growth between two bilayers. Fusion can also be achieved by addition of detergents (usually under 2%) to the liposomal mixture (e.g., Cymal-5™, 1-S-Octyl Beta-D-thioglucopyranoside etc.), incubation with mild agitation and consequent detergent dialysis.

Example 6Proteomics Analysis of hMSCc Ghost and Derived CDLs

[0252]

Method

[0253]

Proteomics analysis was conducted on 4 samples containing ghost cells and CDLs derived from hMSCc that were either conditioned or unconditioned by a medium derived from a prostate cancer cell-line (PC3). For the production of conditioned ghosts and CDLs, hMSCs were incubated for 24 prior to harvesting in medium composed of 50% conditioning media derived from PC3 cells. Cells were then harvested and sonicated ghosts and CDLs were prepared thereof by the method previously described. Sonicated ghosts from conditioned and unconditioned hMSCs (106 cells) were resuspended for analysis in 1 ml TM-buffer, pH 7.4. Cell-derived liposomes derived from 7×106 conditioned and unconditioned hMSCc were resuspended in 50 μL TM buffer, pH 8.6.

[0254]

The samples were sent for proteomics analysis at the proteomics center of the TECHNION—Israel Institute of Technology. Briefly, the samples were digested by Trypsin and the resulting peptides were analyzed by LC-MS/MS. Peptide mix was fractionated by HPLC and electro-sprayed onto an ion-trap mass spectrometer (Orbitrap™). Mass spectrometry was performed in order to analyze the peptides' mass to charge ratio spectra and to determine the proteins' mass. For additional analysis and identification, the peptides were further fragmented by collision induced dissociation (CID) and analyzed again. The peptides were identified by Sequest 3.31 software against the human part of the uniprot database. All protein results are given as Uniport Accession Numbers. The following values were determined for each protein/accession number:

[0000]

MW—Molecular weight
Ppro—The probability of finding a match as good as or better than the observed match by chance. The value displayed for the protein is the probability of the best peptide match (the peptide with the lowest score).
Pep Count—The total number of identified peptides.
Mean—The Average of the peak areas of top 3 identified peptides per protein.
Mean.SE—Mean standard error.
Med—Median of the peak area of all identified peptides per protein
MedErr—Median absolute deviation.

Protein Name.

[0255]

Results

[0256]

The hundreds of proteins that were identified on one or more of the four samples can be divided into 4 distinct groups:

1. Proteins that were prevalent in all four samples (Table 7), i.e. conditioned and unconditioned ghosts and CDLs.

2. Proteins that were prevalent in the ghosts or conditioned ghosts but were missing from the CDLs (Table 5). These proteins are probably or mostly the remains of cytoplasmatic matter that was not completely removed from the ghosts.

3. Proteins that were prevalent only on the conditioned ghosts and CDLs (Table 6). These proteins are probably or mostly membarnal proteins which are only expressed after induction or exposure to condition media.

4. Proteins that were prevalent in all samples but CDLs that were produced from unconditioned hMSCs (Table 7). These proteins are probably or mostly membarnal proteins which are expressed in lower levels on unconditioned hMSCs and which are completely depleted on their derived CDLs. Inducing the hMSCs with condition media probably elevates these proteins level to the extent they become more apparent on the conditioned CDLs.

[0000]

TABLE 4

Proteins prevalent on conditioned and unconditioned ghosts and CDLs.

Fold expression

on conditioned

Uniport

CDLs relative to

Accession

unconditioned

Number

MW

CDLS

Protein name

P04179

24706.6

30

Moesin

P62899

14453.9

26

Myosin regulatory light chain 12A

P09622

54143.1

26

Sodium/potassium-transporting ATPase subunit alpha-1

P02545

74094.8

24

40S ribosomal protein S28

P05023

112824.1

14

Integrin beta-1

P11142

70854.4

14

Cathepsin D

P62701

29579.1

12

Sulfide:quinone oxidoreductase

P07602

58073.9

12

Histone H4

P21589

63327.6

11

Major vault protein

P27797

48111.9

10

Keratin

O75396

24724.8

10

Elongation factor 2

P13674

61011.1

10

Erlin-2

P24752

45170.7

9

60S ribosomal protein L4

P30044

22012.5

9

60S ribosomal protein L18a

P51659

79636.4

9

40S ribosomal protein S21

P61604

10924.9

8

Actin

P17301

129213.8

8

Acetyl-CoA acetyltransferase

P38117

27826.2

8

Leucine-rich PPR motif-containing protein

P10809

61016.5

8

Ras-related protein Rab-7a

P14314

59387.9

7

Dolichyl-diphosphooligosaccharide--protein

glycosyltransferase subunit 2

P62736

41981.8

7

60S ribosomal protein L14

Q96D15

37470

7

Voltage-dependent anion-selective channel protein 3

P62241

24190.2

7

Annexin A4

Q9Y2Q3

25480.3

7

Dolichyl-diphosphooligosaccharide--protein

glycosyltransferase subunit 1

P02786

84818

7

Pyruvate kinase isozymes M1/M2

P38646

73634.8

7

40S ribosomal protein S19

P62829

14856.1

7

Annexin A2

Q9H4B7

50294.6

7

Reticulocalbin-3

P40926

35480.7

7

Peroxiredoxin-1

P08648

114464.9

7

Malate dehydrogenase

P07237

57080.8

7

Prenylcysteine oxidase 1

Q70UQ0

39285

7

40S ribosomal protein S4

Q9NZM1

234558.8

7

Proactivator polypeptide

Q07020

21621.1

7

Heat shock cognate 71 kDa protein

P30040

28975.2

6

Integrin alpha-V

Q09666

628705.2

6

Annexin A11

Q32P28

83341.2

6

60S ribosomal protein L7a

Q15155

134267.4

6

Serine hydroxymethyltransferase

P04040

59718.9

6

Tubulin alpha-1B chain

P30048

27675.2

6

60S ribosomal protein L26-like 1

P68104

50109.2

6

Integrin alpha-2

Q00325

40068.8

6

Myoferlin

P30443

40820.2

6

Trifunctional enzyme subunit beta

P06756

115964.5

6

Thioredoxin-dependent peroxide reductase

Q15149

531465.9

6

ATP synthase subunit O

P16615

114682.7

6

Elongation factor Tu

P14625

92411.2

6

60S ribosomal protein L11

P19105

19781.5

6

60S acidic ribosomal protein P0-like

P34897

55957.8

6

40S ribosomal protein S3

P45880

31546.5

6

Signal recognition particle receptor subunit beta

O15118

142073.5

6

Serpin H1

P62805

11360.4

6

Isocitrate dehydrogenase [NADP]

Q99536

41893.5

6

Endoplasmin

P36957

48698.6

6

Tubulin beta chain

P02751

262439.5

5

CD44 antigen

P13639

95277.1

5

60S ribosomal protein L30

P49411

49510.2

5

60S ribosomal protein L12

Q00610

191491.7

5

Plectin-1

P55072

89265.9

5

Aldehyde dehydrogenase X

P21281

56465

5

ATP synthase subunit alpha

Q16698

36044.8

5

Collagen alpha-1(I) chain

P26038

67777.9

5

40S ribosomal protein S8

P14854

10185.7

5

ATP synthase subunit d

P50454

46411.3

5

Erlin-1

P08670

53619.2

5

Vesicle-trafficking protein SEC22b

Q13423

113822.9

5

Procollagen-lysine

P62847

15413.4

5

Synaptic vesicle membrane protein VAT-1 homolog

P00505

47445.3

5

Protein disulfide-isomerase A6

P05556

88357

5

Niemann-Pick C1 protein

P30837

57202.3

5

Annexin A1

P13667

72887.1

5

Cytochrome c oxidase subunit 6B1

P06733

47139.4

5

Voltage-dependent anion-selective channel protein 2

P68363

50119.6

5

10 kDa heat shock protein

Q15084

48091.3

5

Heme oxygenase 1

P04844

69241.1

5

Lysosome membrane protein 2

P36578

47667.5

5

60S ribosomal protein L22

P11021

72288.5

5

Dipeptidyl peptidase 4

O60568

84731.7

5

ATP synthase subunit beta

P04406

36030.4

5

Keratin

O95816

23757.2

5

Calreticulin

Q04837

17249

5

60S acidic ribosomal protein P2

P09601

32798

5

Transitional endoplasmic reticulum ATPase

Q06830

22096.3

5

Lamin-A/C

Q9H9B4

35596.4

5

Protein disulfide-isomerase A3

P09525

35860.1

5

40S ribosomal protein S5

P16070

81503.4

5

Tubulin beta-1 chain

P04075

39395.3

5

Annexin A5

P14649

22749.7

5

Electron transfer flavoprotein subunit beta

Q02809

83497.5

5

Peptidyl-prolyl cis-trans isomerase B

P10606

13686.9

5

Collagen alpha-1(VI) chain

P48735

50876.9

5

Translocon-associated protein subunit delta

P07339

44523.7

5

Keratin

Q9Y6N5

49928.9

5

Peroxisomal multifunctional enzyme type 2

Q7KZF4

101933.6

5

Prohibitin-2

P50213

39566.1

5

Leucine-rich repeat-containing protein 59

Q9UNX3

17245.6

5

Prolyl 3-hydroxylase 1

P14618

57900.2

5

Lysosome-associated membrane glycoprotein 2

Q96AG4

34908.9

5

40S ribosomal protein S18

P07355

38579.8

4

Guanine nucleotide-binding protein G(I)/G(S)/G(T)

subunit beta-1

P61803

12488.6

4

Dihydrolipoyl dehydrogenase

Q13162

30520.8

4

60S ribosomal protein L5

P63220

9105.6

4

Guanine nucleotide-binding protein subunit beta-2-like 1

P62263

16262.5

4

60S ribosomal protein L18

Q14108

54255.6

4

Histone H2B type 1-B

P63244

35054.6

4

5′-nucleotidase

P49748

70345.5

4

ADP/ATP translocase 2

P55084

51261.6

4

Aminopeptidase N

P42704

157804.6

4

L-lactate dehydrogenase A chain

P00338

36665.4

4

Voltage-dependent anion-selective channel protein 1

Q02543

20748.9

4

Glucosidase 2 subunit beta

Q13885

49875

4

Annexin A6

P30101

56746.8

4

ATP synthase subunit delta

P35268

14777.8

4

Peroxiredoxin-5

Q9P2E9

152380

4

Very long-chain specific acyl-CoA dehydrogenase

P62424

29977

4

Prolow-density lipoprotein receptor-related protein 1

P36543

26128.8

4

Inhibitor of nuclear factor kappa-B kinase-interacting

protein

P62857

7836.2

4

40S ribosomal protein S24

P62753

28663

4

Elongation factor 1-alpha 1

Q9UHG3

56603.8

4

Aspartate aminotransferase

P50914

23417

4

60S ribosomal protein L13

P60174

26652.7

4

Adipocyte plasma membrane-associated protein

P27487

88222.5

4

Fibronectin

P62269

17707.9

4

Myosin light chain 6B

P36542

32975.3

4

Transgelin

P39656

50769

4

Staphylococcal nuclease domain-containing protein 1

Q99623

33275.9

4

Sideroflexin-3

P26373

24246.5

4

60S ribosomal protein L31

Q9HDC9

46450.9

4

Phosphate carrier protein

Q71U36

50103.7

4

ADP/ATP translocase 1

P08865

32833.4

4

Lysosome-associated membrane glycoprotein 1

P00387

34212.7

4

Hexokinase-1

Q9Y5M8

29683.8

4

Protein disulfide-isomerase A4

P25705

59713.7

4

Catalase

P23284

23727.5

4

Cytochrome c oxidase subunit 5A

P05387

11657.9

4

40S ribosomal protein SA

P43307

32215.4

4

Isocitrate dehydrogenase [NAD] subunit alpha

P49755

24960

4

2

P04083

38690

4

Tubulin beta-2C chain

P04843

68526.9

4

V-type proton ATPase subunit E 1

P08133

75825.7

4

40S ribosomal protein S6

P68371

49799

4

V-type proton ATPase catalytic subunit A

O75477

38901.4

4

Trifunctional enzyme subunit alpha

P21796

30753.6

4

Ribosome-binding protein 1

P62873

37353

4

Vimentin

P12235

33043.2

4

Protein S100-A11

P20674

16751.7

4

Procollagen-lysine

Q9BWM7

35480.5

4

Mitochondrial inner membrane protein

P23396

26671.4

4

60S ribosomal protein L23

P05141

32874.2

4

60 kDa heat shock protein

P61247

29925.8

3

Alpha-enolase

Q16891

83626.5

3

Transmembrane emp24 domain-containing protein 9

P51571

18986.6

3

Collagen alpha-2(I) chain

O75489

30222.7

3

Endoplasmic reticulum resident protein 29

P31930

52612.5

3

Cytochrome b-c1 complex subunit 1

P48047

23262.7

3

NADH dehydrogenase [ubiquinone] iron-sulfur protein 3

P62888

12775.7

3

Neuroblast differentiation-associated protein AHNAK

P12111

343450.3

3

Prohibitin

Q8NHW5

34342.7

3

40S ribosomal protein S14

P12109

108462

3

V-type proton ATPase subunit B

P30050

17807.5

3

Triosephosphate isomerase

P40939

82947

3

Integrin alpha-5

O75947

18479.5

3

Fructose-bisphosphate aldolase A

P31040

72645.4

3

Calnexin

P19367

102420.2

3

Cytochrome b-c1 complex subunit 2

P30049

17479.2

3

Prolyl 4-hydroxylase subunit alpha-1

P46782

22862.1

3

Transmembrane emp24 domain-containing protein 10

P39019

16050.5

3

Peroxiredoxin-4

Q07065

65982.9

3

Stomatin-like protein 2

P35232

29785.9

3

BAG family molecular chaperone regulator 2

P62913

20239.7

3

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2

P06576

56524.7

3

Clathrin heavy chain 1

P51149

23474.9

3

Collagen alpha-3(VI) chain

P27824

67526

3

60S ribosomal protein L9

P31949

11732.8

3

HLA class I histocompatibility antigen

P35579

226390.6

3

NADH-ubiquinone oxidoreductase 75 kDa subunit

P02452

138826.8

3

Dolichyl-diphosphooligosaccharide--protein

glycosyltransferase 48 kDa subunit

Q01995

22596.4

3

Dolichyl-diphosphooligosaccharide--protein

glycosyltransferase subunit DAD1

Q14764

99266.1

3

Transferrin receptor protein 1

Q9UJZ1

38510.2

3

40S ribosomal protein S3a

P08123

129209.8

3

NADH-cytochrome b5 reductase 3

P50995

54355.1

2

Sideroflexin-1

P11279

44853.9

2

Actin

Q9BVK6

27260.2

2

Myosin-9

P38606

68260.6

2

NAD(P) transhydrogenase

P08758

35914.4

2

60S ribosomal protein L7

P28331

79416.7

2

CD59 glycoprotein

P15144

109470.8

2

Cytochrome c oxidase subunit 5B

P22695

48412.9

2

Glyceraldehyde-3-phosphate dehydrogenase

P35908

65393.2

2

Glutathione S-transferase kappa 1

P35527

62026.7

2

Tubulin alpha-1A chain

Q07954

504243.2

2

Dihydrolipoyllysine-residue succinyltransferase

component of 2-oxoglutarate dehydrogenase complex

P33778

13941.6

2

Succinate dehydrogenase [ubiquinone] flavoprotein subunit

P13645

58791.6

1

Cytoskeleton-associated protein 4

P60709

41709.7

1

Keratin

P46777

34340.7

1

Stress-70 protein

Q9Y277

30639.3

1

Protein disulfide-isomerase

P32969

21849.8

1

ATP synthase subunit gamma

P18124

29207.2

1

Translocon-associated protein subunit alpha

O94905

37815.5

1

Single-stranded DNA-binding protein

P13987

14167.8

1

78 kDa glucose-regulated protein

P07437

49639

1

Nodal modulator 1

P13473

44932.3

1

Superoxide dismutase [Mn]

P04264

65998.9

1

Tubulin beta-2A chain

[0000]

TABLE 5

Proteins that were prevalent in the ghosts or conditioned ghosts but were missing from the CDLs

Uniport

Prevalent in

Accession

Prevalent

conditioned

Number

MW

in ghosts

ghosts

Protein name

P78527

468786.9

+

+

Actin-related protein 2/3 complex subunit 1B

Q99715

332939.7

+

+

rRNA 2′-O-methyltransferase fibrillarin

Q14573

303910.4

+

+

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 8

P24821

240697.7

+

+

E3 ubiquitin-protein ligase MARCH5

P35580

228856.9

+

+

Acyl carrier protein

P07942

197935.7

+

+

Fibrillin-1

P55268

195853.3

+

+

Extracellular sulfatase Sulf-1

Q8WWI1

192589.5

+

+

Protein kinase C alpha type

P20908

183446.3

+

+

Cytosol aminopeptidase

P11047

177487.9

+

+

Beta-galactosidase

Q6YHK3

161586.8

+

+

Histone H3.1t

Q7L576

145088.6

+

+

GDP-fucose protein O-fucosyltransferase 1

Q08211

140868.9

+

+

Protein S100-A6

P23634

137832.9

+

+

Protein tyrosine phosphatase-like protein PTPLAD1

Q12768

134200.9

+

+

39S ribosomal protein L43

P00533

134190.2

+

+

Radixin

O43795

131901.9

+

+

Putative ribosomal RNA methyltransferase NOP2

Q9BSJ8

122780.1

+

+

Trophoblast glycoprotein

P26006

118680.2

+

+

Galectin-3

P54707

115437.2

+

+

ATP synthase subunit f

Q8N766

111689.2

+

+

Calponin-2

O60313

111560.7

+

+

Tumor necrosis factor receptor superfamily member 10B

Q9Y4L1

111266.2

+

+

Probable glutathione peroxidase 8

Q6P179

110391.1

+

+

Hydroxyacyl-coenzyme A dehydrogenase

P22413

104856.7

+

+

Medium-chain specific acyl-CoA dehydrogenase

Q6ZXV5

103941.9

+

+

SH3 domain-binding glutamic acid-rich-like protein 3

A0FGR8

102294.1

+

+

V-type proton ATPase 116 kDa subunit a isoform 3

P11586

101495.6

+

+

U5 small nuclear ribonucleoprotein 200 kDa helicase

Q15063

93255.4

+

+

Transducin beta-like protein 2

Q13488

92908.6

+

+

Extended synaptotagmin-2

O95479

88836.6

+

+

Mitochondrial import inner membrane translocase subunit

Tim8 A

Q9UBV2

88698.6

+

+

Histone H1.2

Q9NR30

87290.5

+

+

Vesicular integral-membrane protein VIP36

Q15436

86105.3

+

+

SRA stem-loop-interacting RNA-binding protein

Q99798

85372

+

+

Nuclear pore complex protein Nup205

P08238

83212.2

+

+

DnaJ homolog subfamily B member 1

P13010

82652.4

+

+

HEAT repeat-containing protein 1

Q96TA1

82631.1

+

+

Pyruvate dehydrogenase E1 component subunit alpha

Q8IVL6

81785.8

+

+

Pre-mRNA-processing-splicing factor 8

Q9UH99

80261.7

+

+

Collagen alpha-1(XIV) chain

Q9BU23

79647.6

+

+

Probable saccharopine dehydrogenase

Q96AC1

77810.7

+

+

Nucleoside diphosphate kinase B

P21980

77279.8

+

+

Protein DEK

Q6NUQ4

77101.6

+

+

Nascent polypeptide-associated complex subunit alpha

P17252

76714.3

+

+

LIM domain only protein 7

P23246

76101.8

+

+

NADH dehydrogenase [ubiquinone] iron-sulfur protein 6

Q99805

75725.7

+

+

Peptidyl-tRNA hydrolase 2

O75746

74715

+

+

Deoxyribonuclease-2-alpha

P46063

73410

+

+

Alpha-L-iduronidase

Q8NBJ5

71590.6

+

+

Cytochrome c oxidase subunit 6C

P17066

70984.4

+

+

Signal peptidase complex subunit 2

O43390

70899.2

+

+

60S ribosomal protein L35

P43155

70812.5

+

+

Adenosine 3′-phospho 5′-phosphosulfate transporter 1

P34931

70331.5

+

+

Proliferation-associated protein 2G4

P54652

69978

+

+

Splicing factor

P17844

69104.8

+

+

Ras-related protein Ral-A

Q96CM8

68080.8

+

+

60S ribosomal protein L10-like

Q03252

67647.6

+

+

ATP-binding cassette sub-family E member 1

O94826

67412.2

+

+

Actin-related protein 2/3 complex subunit 2

P20700

66367.7

+

+

Cathepsin Z

Q5JTV8

66208.4

+

+

Acyl-coenzyme A thioesterase 1

O00567

66008.8

+

+

Signal peptidase complex catalytic subunit SEC11A

P23368

65402

+

+

Acetyl-coenzyme A transporter 1

Q10471

64691.5

+

+

4F2 cell-surface antigen heavy chain

Q10472

64177.5

+

+

Tropomyosin beta chain

P14866

64092.4

+

+

Coiled-coil domain-containing protein 47

Q14956

63882

+

+

Myb-binding protein 1A

P07686

63071.3

+

+

ADP-ribosylation factor 1

O95302

63043.6

+

+

Synaptonemal complex protein SC65

Q969V3

62934.7

+

+

Signal peptidase complex subunit 3

P30038

61680.7

+

+

NADH dehydrogenase [ubiquinone] iron-sulfur protein 2

Q96S52

61617.3

+

+

Ras-related protein Rab-8A

Q9HCC0

61294.5

+

+

EH domain-containing protein 2

Q5SSJ5

61169.3

+

+

Rho GTPase-activating protein 1

Q9P0J1

61015.7

+

+

Putative 40S ribosomal protein S26-like 1

Q9H857

60679.8

+

+

Mitochondrial chaperone BCS1

P04062

59678.4

+

+

Calcium-binding mitochondrial carrier protein Aralar2

Q9Y2X3

59540.6

+

+

Ribosome-releasing factor 2

Q7Z4H8

58535.3

+

+

39S ribosomal protein L46

Q13217

57544.3

+

+

Cytochrome c1

P49257

57513.1

+

+

Transmembrane 9 superfamily member 4

P26599

57185.8

+

+

60S ribosomal protein L36a

P05091

56345.7

+

+

Metaxin-2

Q96A33

55838.4

+

+

Heterogeneous nuclear ribonucleoprotein L

Q9UMS4

55146.4

+

+

ES1 protein homolog

O60701

54989.3

+

+

Replication protein A 14 kDa subunit

O76021

54939

+

+

60S ribosomal protein L37a

P10619

54431.2

+

+

Neuron-specific calcium-binding protein hippocalcin

Q96HE7

54358.1

+

+

Translocation protein SEC63 homolog

Q15233

54197.4

+

+

Protein transport protein Sec61 subunit beta

Q02818

53846.4

+

+

Torsin-1A-interacting protein 2

P22570

53803.1

+

+

NADH dehydrogenase [ubiquinone] iron-sulfur protein 7

Q96N66

52730.3

+

+

Fatty aldehyde dehydrogenase

P20073

52705.8

+

+

All-trans-retinol 13

P61619

52230.6

+

+

H/ACA ribonucleoprotein complex subunit 4

Q9Y512

51943.4

+

+

Multidrug resistance-associated protein 1

O43615

51323.6

+

+

Neuroplastin

Q07960

50404.3

+

+

Heterochromatin protein 1-binding protein 3

Q13509

50400.3

+

+

Transmembrane emp24 domain-containing protein 3

Q9P2R7

50285.3

+

+

Phosphoglycerate mutase 1

P80303

50164.4

+

+

Putative heterogeneous nuclear ribonucleoprotein A1-like 3

P36551

50120.1

+

+

Isovaleryl-CoA dehydrogenase

P13489

49941.2

+

+

ATP synthase subunit b

Q9Y305

49869.6

+

+

Ras-related protein Rab-5A

Q9BUF5

49825

+

+

Poly(rC)-binding protein 2

P04350

49553.9

+

+

Actin-related protein 2/3 complex subunit 5

P31943

49198.4

+

+

60S ribosomal protein L13a

P62495

49000.2

+

+

Mitochondrial import inner membrane translocase subunit

Tim13

P82675

47976.2

+

+

Beta-actin-like protein 2

P09543

47548.7

+

+

Protein disulfide-isomerase TMX3

P45954

47455.3

+

+

Acid ceramidase

Q8NBX0

47121.5

+

+

Lipase maturation factor 2

O60664

47018

+

+

Ras-related C3 botulinum toxin substrate 1

P28300

46914.5

+

+

Golgin subfamily B member 1

P11310

46558.6

+

+

Ectonucleotide pyrophosphatase/phosphodiesterase

family member 1

O75718

46532

+

+

Ras-related protein Rab-18

O14979

46409

+

+

High mobility group protein B1

P26440

46289.7

+

+

Coproporphyrinogen-III oxidase

P60842

46124.6

+

+

Contactin-associated protein 1

Q58FF3

45829.9

+

+

Protein S100-A16

Q96HD1

45408.9

+

+

Isocitrate dehydrogenase [NAD] subunit beta

Q6YN16

45365.5

+

+

CDP-diacylglycerol--inositol 3-phosphatidyltransferase

Q8NC51

44938.5

+

+

Follistatin-related protein 1

Q96G23

44847.4

+

+

Ribose-phosphate pyrophosphokinase 1

Q9BTV4

44847.3

+

+

Eukaryotic initiation factor 4A-III

P61160

44732.3

+

+

Probable cation-transporting ATPase 13A1

P09110

44263.9

+

+

High mobility group protein HMGI-C

P07093

43974.3

+

+

Mesoderm-specific transcript homolog protein

Q9H488

43927.2

+

+

Cytoplasmic FMR1-interacting protein 1

O75521

43557.4

+

+

Matrin-3

Q6NVY1

43454.4

+

+

Polypeptide N-acetylgalactosaminyltransferase 1

P17302

42980.9

+

+

39S ribosomal protein L1

Q13336

42499.8

+

+

Dolichol-phosphate mannosyltransferase

Q16795

42482.6

+

+

Peptidyl-prolyl cis-trans isomerase C

P35613

42174.1

+

+

Gamma-glutamyl hydrolase

P29992

42096.6

+

+

V-type proton ATPase 16 kDa proteolipid subunit

Q9BYX7

41988.9

+

+

Ribosome biogenesis regulatory protein homolog

Q562R1

41976

+

+

28S ribosomal protein S5

Q9BRK5

41780.5

+

+

Lanosterol synthase

P30533

41440.9

+

+

Glyoxylate reductase/hydroxypyruvate reductase

P82650

41254.4

+

+

Transforming growth factor-beta-induced protein ig-h3

Q15050

41168.2

+

+

Beta-actin-like protein 3

Q12907

40203.1

+

+

Nucleolar RNA helicase 2

O75367

39592.5

+

+

CAAX prenyl protease 1 homolog

Q9NYL9

39570.3

+

+

RNA-binding protein FUS

P09972

39431.3

+

+

ADP-ribosylation factor-like protein 6-interacting protein 1

Q5EB52

38805.5

+

+

Glia-derived nexin

Q15366

38555.6

+

+

Tubulin beta-6 chain

Q9H0U3

38011.4

+

+

Magnesium transporter protein 1

O15121

37841.1

+

+

60S ribosomal protein L6

Q9UDY4

37783.2

+

+

Peptidyl-prolyl cis-trans isomerase FKBP3

P62136

37487.8

+

+

Up-regulated during skeletal muscle growth protein 5

Q14257

36853.7

+

+

Thioredoxin-related transmembrane protein 1

P05198

36089.4

+

+

28S ribosomal protein S31

Q9NZ01

36010.8

+

+

High mobility group protein HMG-I/HMG-Y

P27695

35532.2

+

+

Antigen peptide transporter 1

P08574

35367

+

+

DnaJ homolog subfamily C member 3

P31937

35305.8

+

+

Peroxisomal acyl-coenzyme A oxidase 1

Q08257

35184.6

+

+

Chloride intracellular channel protein 1

Q15006

34811.4

+

+

N-acetylglucosamine-6-sulfatase

P09486

34609.7

+

+

Probable transcription factor PML

O15144

34311.5

+

+

Mitochondrial import receptor subunit TOM70

Q16836

34255.9

+

+

Endoplasmic reticulum aminopeptidase 2

Q9UHQ9

34073.2

+

+

LDLR chaperone MESD

P53007

33991

+

+

Acyl-coenzyme A thioesterase 13

Q9UBR2

33846.2

+

+

Lamin-B1

Q8NBJ7

33835.8

+

+

116 kDa U5 small nuclear ribonucleoprotein component

P62995

33645.6

+

+

Proteolipid protein 2

Q9BPW8

33288.9

+

+

Collagen triple helix repeat-containing protein 1

P07951

32830.6

+

+

Isocitrate dehydrogenase [NAD] subunit gamma

P42126

32795.2

+

+

RNA-binding Raly-like protein

Q02878

32707.6

+

+

Sphingolipid delta(4)-desaturase DES1

Q86SE5

32310.6

+

+

3

Q9Y639

31271.9

+

+

Mitochondrial import inner membrane translocase subunit

TIM44

P15559

30848

+

+

Mammalian ependymin-related protein 1

O75431

29744.1

+

+

Aldehyde dehydrogenase

O60762

29615.8

+

+

Urea transporter 1

P22090

29437

+

+

Retinol dehydrogenase 11

P62258

29155.4

+

+

T-complex protein 1 subunit delta

P24539

28890.3

+

+

Ribonuclease inhibitor

P18669

28785.9

+

+

Splicing factor

P67936

28504.5

+

+

60S ribosomal protein L28

Q9UFN0

28448.5

+

+

Calpain small subunit 1

Q9UHQ4

28302.2

+

+

Histone H1.1

P30042

28152.7

+

+

Pre-mRNA-processing factor 19

P57088

27959.8

+

+

NADH dehydrogenase [ubiquinone] iron-sulfur protein 5

Q9P0L0

27875.2

+

+

OCIA domain-containing protein 2

Q07955

27727.8

+

+

Histone H1.0

P63104

27727.7

+

+

Glycylpeptide N-tetradecanoyltransferase 1

Q9NR28

27113.7

+

+

Putative 60S ribosomal protein L13a-like MGC87657

P33316

26689.7

+

+

Major facilitator superfamily domain-containing protein

10

O75352

26620.5

+

+

Transmembrane emp24 domain-containing protein 1

P54819

26460.8

+

+

Cystatin-B

P17931

26136.1

+

+

Integrin alpha-3

P60033

25792.1

+

+

Signal peptidase complex subunit 1

Q9UM22

25420.6

+

+

NAD(P)H dehydrogenase [quinone] 1

Q13445

25189.7

+

+

Mannose-P-dolichol utilization defect 1 protein

Q00688

25161.3

+

+

DnaJ homolog subfamily B member 4

Q15005

24986.7

+

+

Heat shock 70 kDa protein 6

P09429

24878.2

+

+

Heterogeneous nuclear ribonucleoprotein D-like

P62906

24815.5

+

+

Protein Mpv17

Q9Y3Q3

24761.3

+

+

Tubulin beta-3 chain

P27635

24587.9

+

+

Anoctamin-10

Q96L21

24502.7

+

+

Acyl-CoA synthetase family member 2

P62826

24407.6

+

+

Heat shock protein beta-1

B2RPK0

24222.8

+

+

Peptidyl-prolyl cis-trans isomerase FKBP7

O43402

23757.7

+

+

Thioredoxin reductase 2

P20339

23643.8

+

+

Acyl-coenzyme A thioesterase 9

P40429

23562.4

+

+

Heterogeneous nuclear ribonucleoprotein H

P11233

23552

+

+

Probable ATP-dependent RNA helicase DDX5

O14735

23523.1

+

+

Hydroxysteroid dehydrogenase-like protein 2

Q8N983

23416.2

+

+

WASH complex subunit strumpellin

P45877

22748.8

+

+

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 9

P46781

22577.6

+

+

Nucleosome assembly protein 1-like 1

P84074

22413

+

+

Lysosomal protective protein

Q96AB3

22322.8

+

+

Beta-hexosaminidase subunit alpha

O43399

22224.3

+

+

Serum albumin

Q02539

21828.9

+

+

B-cell receptor-associated protein 29

O75915

21600.4

+

+

Mitochondrial import inner membrane translocase subunit

TIM50

Q9H061

21513.5

+

+

NADH dehydrogenase [ubiquinone] flavoprotein 2

P16403

21351.8

+

+

Protein sel-1 homolog 1

P84077

20683.7

+

+

Beta-hexosaminidase subunit beta

P67812

20612.1

+

+

Nucleolar protein 56

P18085

20497.7

+

+

Sterol-4-alpha-carboxylate 3-dehydrogenase

P24844

19814.5

+

+

WASH complex subunit 7

O60831

19245.5

+

+

Glycine cleavage system H protein

P46778

18553.1

+

+

Putative hexokinase HKDC1

P62280

18419

+

+

SWI/SNF complex subunit SMARCC2

P62277

17211.7

+

+

39S ribosomal protein L38

Q9BX68

17151.2

+

+

Ganglioside GM2 activator

P15531

17137.7

+

+

Oligosaccharyltransferase complex subunit OSTC

P62841

17029.2

+

+

Small nuclear ribonucleoprotein-associated proteins B

and B′

P63241

16821.4

+

+

Regulator of chromosome condensation

Q9NRP0

16817.8

+

+

Nucleoside diphosphate kinase A

Q86SX6

16617.5

+

+

60S ribosomal protein L34

O15511

16310.3

+

+

Tubulin beta-4 chain

P46779

15737.7

+

+

Tropomyosin alpha-4 chain

P26885

15639.3

+

+

Receptor expression-enhancing protein 5

O60361

15519

+

+

Putative pre-mRNA-splicing factor ATP-dependent RNA

helicase DHX15

Q9NS69

15511.8

+

+

Four and a half LIM domains protein 2

Q9Y3E0

15415.4

+

+

ATP-dependent RNA helicase DHX29

P69905

15247.9

+

+

Leucyl-cystinyl aminopeptidase

P42766

14542.6

+

+

Heterogeneous nuclear ribonucleoprotein R

P55769

14164.6

+

+

Histidyl-tRNA synthetase

P04908

14127

+

+

Small nuclear ribonucleoprotein Sm D3

P61769

13705.9

+

+

Prostacyclin synthase

Q5JNZ5

12994

+

+

[Pyruvate dehydrogenase [acetyl-transferring]]-

phosphatase 1

Q9GZT3

12341.4

+

+

Protein transport protein Sec23A

P84090

12251

+

+

Abhydrolase domain-containing protein 10

Q96FQ6

11794

+

+

Putative endoplasmin-like protein

P99999

11741.1

+

+

60S acidic ribosomal protein P1

P17096

11669.2

+

+

Trans-2

O60220

10991.3

+

+

GDH/6PGL endoplasmic bifunctional protein

P56134

10910.7

+

+

Potassium-transporting ATPase alpha chain 2

Q9Y5L4

10493

+

+

Eukaryotic peptide chain release factor subunit 1

Q9H299

10431.3

+

+

Transmembrane and TPR repeat-containing protein 3

P61513

10268.5

+

+

Ribosomal L1 domain-containing protein 1

O75531

10052

+

+

Regulator of microtubule dynamics protein 1

P60468

9968.1

+

+

Adenylate kinase 2

P62979

9411.9

+

+

Alpha-2-macroglobulin

P63173

8212.7

+

+

72 kDa type IV collagenase

P04732

6009.2

+

+

SUN domain-containing protein 1

P35555

312082

+

−

Laminin subunit beta-1

Q05707

193393

+

−

SUN domain-containing protein 2

Q7Z478

155138.3

+

−

Vesicle transport protein GOT1B

Q9HD20

132869.9

+

−

Actin-related protein 2

Q9UIQ6

117274.2

+

−

Hemoglobin subunit alpha

Q9UGP8

87941.5

+

−

ERO1-like protein alpha

Q9UJS0

74128.8

+

−

Glucosylceramidase

P27658

73317.2

+

−

Metaxin-1

P35475

72624.8

+

−

ATP-dependent DNA helicase Q1

P02768

69321.6

+

−

Tumor protein D54

P35241

68521.5

+

−

Epidermal growth factor receptor

P08195

67951.9

+

−

Polypeptide N-acetylgalactosaminyltransferase 2

Q6NUM9

66776.8

+

−

Annexin A7

Q9NNW7

66320.8

+

−

Neighbor of COX4

Q86UE4

63798.8

+

−

Actin-related protein 2/3 complex subunit 3

P12081

57374.3

+

−

NHP2-like protein 1

Q9UBM7

54453.8

+

−

Cleft lip and palate transmembrane protein 1

P49189

53767.1

+

−

ATP-binding cassette sub-family D member 3

O15269

52710.6

+

−

Arylsulfatase A

O75306

52511.8

+

−

Delta-1-pyrroline-5-carboxylate dehydrogenase

P12694

50439.2

+

−

ADP-dependent glucokinase

Q92791

50349.3

+

−

Peptidyl-prolyl cis-trans isomerase FKBP9

Q15113

47942

+

−

Prostaglandin E synthase 2

Q8TB61

47483.6

+

−

Carnitine O-acetyltransferase

P55209

45346

+

−

40S ribosomal protein S9

Q92665

45290.5

+

−

Eukaryotic translation initiation factor 2 subunit 1

Q96DV4

44568.4

+

−

40S ribosomal protein S13

Q13561

44203.9

+

−

Coiled-coil domain-containing protein 109A

Q9UQ80

43759.2

+

−

Heat shock 70 kDa protein 1-like

P29803

42905.6

+

−

Niban-like protein 1

P51553

42767.1

+

−

3-hydroxyisobutyryl-CoA hydrolase

P61163

42586.9

+

−

Splicing factor

P25685

38020.4

+

−

Heat shock protein HSP 90-beta

Q9H2U2

37896

+

−

CD166 antigen

Q9H7B2

35560.2

+

−

Transient receptor potential cation channel subfamily V

member 2

Q8TC12

35363.5

+

−

40S ribosomal protein S4

Q12841

34962.9

+

−

Plasminogen activator inhibitor 1 RNA-binding protein

P60891

34811.9

+

−

LAG1 longevity assurance homolog 2

Q99439

33675.3

+

−

Uncharacterized protein KIAA0090

Q86WA6

32522

+

−

Putative phospholipase B-like 2

Q14192

32170.6

+

−

Mitochondrial import receptor subunit TOM22 homolog

P16152

30355.9

+

−

28S ribosomal protein S23

Q9Y680

29990.1

+

−

Putative high mobility group protein B1-like 1

O60613

27629

+

−

ATP-dependent RNA helicase DDX18

Q96CG8

26207.1

+

−

Protein NipSnap homolog 1

Q14696

26060.3

+

−

NADH-cytochrome b5 reductase 1

Q13765

23369.7

+

−

Transmembrane protein 214

P09211

23341

+

−

40S ribosomal protein S29

P04792

22768.5

+

−

GTP-binding nuclear protein Ran

Q9Y3D9

21757.3

+

−

Carbonyl reductase [NADPH] 1

Q00765

21479.1

+

−

Peptidyl-prolyl cis-trans isomerase FKBP2

P63000

21436.3

+

−

Perilipin-3

P05976

21131.8

+

−

Nuclear pore complex protein Nup155

P30086

21043.7

+

−

Vesicle-associated membrane protein 2

Q9ULC4

20541.8

+

−

Succinyl-CoA ligase [ADP-forming] subunit beta

P61009

20300.5

+

−

Nicalin

P51970

20092.1

+

−

Inositol 1

P84103

19317.9

+

−

Malignant T cell-amplified sequence 1

Q9Y6H1

15502.7

+

−

U1 small nuclear ribonucleoprotein C

Q9NPJ3

14950.9

+

−

Tricarboxylate transport protein

P35244

13559.9

+

−

UDP-glucose 6-dehydrogenase

O43920

12509.4

+

−

Transmembrane protein 33

Q6NVV1

12126.9

+

−

Diablo homolog

P04080

11132.6

+

−

Non-POU domain-containing octamer-binding protein

O43678

10914.8

+

−

Ankycorbin

P09669

8775.7

+

−

Procollagen galactosyltransferase 1

Q15738

NA

−

+

ADP-ribosylation factor 4

P17900

NA

−

+

Histidine triad nucleotide-binding protein 2

P46087

NA

−

+

Myosin-Ib

O00299

NA

−

+

Quinone oxidoreductase

P39210

NA

−

+

60S ribosomal protein L10a

Q9P035

NA

−

+

Plasma membrane calcium-transporting ATPase 4

Q9NUJ1

NA

−

+

Enhancer of rudimentary homolog

O43837

NA

−

+

Cysteine-rich with EGF-like domain protein 1

Q9Y5S1

NA

−

+

Ribosome production factor 2 homolog

Q16647

NA

−

+

Beta-2-microglobulin

Q96JJ7

NA

−

+

2′

P23434

NA

−

+

PRA1 family protein 2

POC7M2

NA

−

+

Nucleobindin-2

P15289

NA

−

+

Serine palmitoyltransferase 1

O75844

NA

−

+

Core histone macro-H2A.1

P09234

NA

−

+

Coiled-coil-helix-coiled-coil-helix domain-containing

protein 2

P08253

NA

−

+

60S ribosomal protein L38

P04632

NA

−

+

Protein NipSnap homolog 3A

Q03518

NA

−

+

DNA-(apurinic or apyrimidinic site) lyase

Q8NFQ8

NA

−

+

Nucleobindin-1

P07305

NA

−

+

Splicing factor

Q15029

NA

−

+

Sulfatase-modifying factor 2

O75643

NA

−

+

C-1-tetrahydrofolate synthase

Q9Y276

NA

−

+

5′-nucleotidase domain-containing protein 2

Q9BYD6

NA

−

+

Gap junction alpha-1 protein

Q8IWU6

NA

−

+

Laminin subunit beta-2

Q9UBQ7

NA

−

+

Alpha-2-macroglobulin receptor-associated protein

P61221

NA

−

+

Lamin-B2

P63027

NA

−

+

Periostin

Q9Y4P3

NA

−

+

Phosphatidylethanolamine-binding protein 1

P30419

NA

−

+

14-3-3 protein zeta/delta

P50991

NA

−

+

14-3-3 protein epsilon

O94901

NA

−

+

Metallothionein-1E

Q9H7Z7

NA

−

+

Procollagen C-endopeptidase enhancer 1

P22087

NA

−

+

Collagen alpha-1(XII) chain

P28288

NA

−

+

4-trimethylaminobutyraldehyde dehydrogenase

Q13505

NA

−

+

Collagen alpha-1(VIII) chain

Q9Y3E5

NA

−

+

Transmembrane 9 superfamily member 2

P27449

NA

−

+

Guanine nucleotide-binding protein subunit alpha-11

Q13510

NA

−

+

Short/branched chain specific acyl-CoA dehydrogenase

P62318

NA

−

+

Histone H2A type 1-B/E

O00115

NA

−

+

Calcium-binding mitochondrial carrier protein Aralar1

P33527

NA

−

+

Sorting and assembly machinery component 50 homolog

Q8NE86

NA

−

+

Dynactin subunit 2

O60832

NA

−

+

Protein transport protein Sec61 subunit alpha isoform 1

Q15582

NA

−

+

28S ribosomal protein S22

Q13740

NA

−

+

Inorganic pyrophosphatase 2

Q9NVP1

NA

−

+

15 kDa selenoprotein

P22392

NA

−

+

Fermitin family homolog 2

P43243

NA

−

+

Peroxisomal 3

P38919

NA

−

+

Transmembrane protein 43

Q04941

NA

−

+

Transformer-2 protein homolog beta

P05386

NA

−

+

Cytochrome c

Q8NHP8

NA

−

+

40S ribosomal protein S11

Q8TAQ2

NA

−

+

Valacyclovir hydrolase

O15145

NA

−

+

Protein LYRIC

Q86TX2

NA

−

+

Torsin-1A-interacting protein 1

Q16629

NA

−

+

Alpha-centractin

P35637

NA

−

+

Tropomodulin-3

Q9NX47

NA

−

+

Cartilage-associated protein

Q9NP72

NA

−

+

Tenascin

Q9NZN4

NA

−

+

Methylcrotonoyl-CoA carboxylase beta chain

P51648

NA

−

+

Lysophospholipid acyltransferase 7

O15143

NA

−

+

DNA-dependent protein kinase catalytic subunit

Q92820

NA

−

+

Basigin

Q9Y6A9

NA

−

+

CD81 antigen

P29590

NA

−

+

SPARC

Q6P2Q9

NA

−

+

Prolyl 3-hydroxylase 3

Q13641

NA

−

+

60S ribosomal protein L21

Q2TB90

NA

−

+

Extended synaptotagmin-1

Q14789

NA

−

+

Protein-lysine 6-oxidase

P15586

NA

−

+

Tetratricopeptide repeat protein 35

O75694

NA

−

+

Myosin light chain 1/3

Q56VL3

NA

−

+

Vesicle-associated membrane protein-associated protein A

P61006

NA

−

+

GPI transamidase component PIG-S

P49207

NA

−

+

Glutaredoxin-related protein 5

P14678

NA

−

+

40S ribosomal protein S15

Q96IX5

NA

−

+

Serine/threonine-protein phosphatase PP1-alpha catalytic

subunit

O75380

NA

−

+

Splicing factor

Q969S9

NA

−

+

Nucleolar protein 58

P52926

NA

−

+

3-ketoacyl-CoA thiolase

Q9H2W6

NA

−

+

KDEL motif-containing protein 2

Q08170

NA

−

+

Heat shock-related 70 kDa protein 2

O14763

NA

−

+

Dynamin-like 120 kDa protein

Q15067

NA

−

+

3-hydroxyisobutyrate dehydrogenase

Q2M389

NA

−

+

Myosin regulatory light polypeptide 9

Q3ZCQ8

NA

−

+

PRA1 family protein 3

Q9BRR6

NA

−

+

2-oxoisovalerate dehydrogenase subunit alpha

P78357

NA

−

+

Eukaryotic initiation factor 4A-I

P19404

NA

−

+

Transmembrane protein 126A

P35659

NA

−

+

Protein-glutamine gamma-glutamyltransferase 2

P16278

NA

−

+

Laminin subunit gamma-1

Q9P0K7

NA

−

+

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 2

P18754

NA

−

+

Eukaryotic translation initiation factor 5A-1

P62273

NA

−

+

Glutathione S-transferase P

Q14728

NA

−

+

Deoxyuridine 5′-triphosphate nucleotidohydrolase

O75251

NA

−

+

Reticulocalbin-2

Q9H3N1

NA

−

+

NADPH:adrenodoxin oxidoreductase

P06703

NA

−

+

ATP-dependent RNA helicase A

O96005

NA

−

+

7-dehydrocholesterol reductase

P28838

NA

−

+

Collagen alpha-1(V) chain

P83881

NA

−

+

Polypyrimidine tract-binding protein 1

O43143

NA

−

+

Putative nucleoside diphosphate kinase

O00400

NA

−

+

NAD-dependent malic enzyme

Q8TED1

NA

−

+

Hypoxia up-regulated protein 1

Q9H583

NA

−

+

X-ray repair cross-complementing protein 5

Q16695

NA

−

+

CD109 antigen

Q96DB5

NA

−

+

Barrier-to-autointegration factor

Q9NW15

NA

−

+

60S ribosomal protein L10

P01023

NA

−

+

40S ribosomal protein S27a

Q92544

NA

−

+

Protein ERGIC-53

Q9BQG0

NA

−

+

Transmembrane glycoprotein NMB

P48449

NA

−

+

Fructose-bisphosphate aldolase C

P06865

NA

−

+

45 kDa calcium-binding protein

O14561

NA

−

+

Isochorismatase domain-containing protein 2

Q15041

NA

−

+

Aconitate hydratase

Q92621

NA

−

+

Myosin-10

[0000]

TABLE 6

Proteins that were prevalent only on the conditioned ghosts and CDLs

Ratio of

expression:

conditioned

Uniport

CDLs/

Accession

conditioned

Number

MW

ghost

Protein name

Q15717

NA

100%

Endoplasmic reticulum lectin 1

P14136

NA

100%

Signal recognition particle receptor

subunit alpha

Q32P51

NA

17%

Long-chain-fatty-acid--CoA ligase 3

Q9NVA2

NA

16%

Cation-independent mannose-6-

phosphate receptor

O75131

NA

11%

Granulins

Q96DZ1

NA

10%

Heterogeneous nuclear

ribonucleoprotein K

P01889

NA

9%

DnaJ homolog subfamily C member 10

Q16270

NA

9%

Copine-3

P61978

NA

7%

Flotillin-1

P21912

NA

6%

Metalloproteinase inhibitor 3

P28799

NA

6%

Glial fibrillary acidic protein

Q8IXB1

NA

6%

Translational activator GCN1

Q5KU26

NA

5%

Heterogeneous nuclear

ribonucleoprotein A1-like 2

O75955

NA

4%

Protein FAM98A

P35625

NA

4%

Septin-11

Q92616

NA

4%

Succinate dehydrogenase [ubiquinone]

iron-sulfur subunit

O95573

NA

3%

Collectin-12

Q8NCA5

NA

3%

Insulin-like growth factor-binding

protein 7

P11717

NA

2%

HLA class I histocompatibility antigen

[0000]

TABLE 7

Proteins that were prevalent in ghosts, conditioned ghosts and conditioned CDLs but were missing from

unconditioned CDLs.

Ratio of expression

Uniport

Cond

Cond.

Accession

Ghosts/

CDLs/Cond.

Number

MW

Ghosts

Ghosts

Protein name

Q53EP0

132803.2

302%

2%

Calumenin

P52272

77464.3

262%

2%

Nucleophosmin

P12956

69799.2

248%

1%

Cytochrome c oxidase subunit 2

O60716

108103.3

220%

4%

Glutaminase kidney isoform

P05121

45031.1

218%

5%

Integrin beta-5

P18621

21383.3

218%

13%

Plasma membrane calcium-transporting ATPase 1

Q9H845

68716.8

209%

7%

Nucleolin

Q14103

38410.3

198%

0%

60S acidic ribosomal protein P0

P61916

16559.5

195%

2%

Atlastin-3

Q02978

34039.9

193%

3%

NADH dehydrogenase [ubiquinone] iron-sulfur protein 8

Q969X5

32571.5

186%

8%

Neutral alpha-glucosidase AB

P07858

37796.8

179%

2%

Cytochrome c oxidase subunit 4 isoform 1

O95831

66859

176%

4%

Complement component 1 Q subcomponent-binding protein

P51148

23467.8

161%

6%

3-hydroxyacyl-CoA dehydrogenase type-2

Q9H0U4

22157.2

161%

1%

AP-2 complex subunit alpha-1

O75390

51679.6

157%

3%

Adenylyl cyclase-associated protein 1

Q8TCJ2

93613.8

155%

1%

Collagen alpha-1(III) chain

P21964

30017.6

148%

0%

Mitochondrial carrier homolog 2

O43852

37083.6

143%

2%

Fibronectin type III domain-containing protein 3B

O95782

107477.9

142%

5%

Ras-related protein Rab-1B

P50416

88310.8

142%

2%

Alpha-actinin-1

Q9NYU2

177077.4

140%

1%

Hydroxymethylglutaryl-CoA lyase

P46977

80476.9

138%

3%

40S ribosomal protein S12

P49821

50784.9

137%

5%

Actin-related protein 3

Q12797

85809.5

135%

5%

40S ribosomal protein S20

P46940

189132.9

133%

1%

Thy-1 membrane glycoprotein

Q9ULV4

53215.1

131%

3%

Coronin-1C

O00159

121648.1

129%

4%

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 10

P07996

129299.2

129%

2%

Mitochondrial-processing peptidase subunit beta

O14773

61209.7

129%

6%

Mesencephalic astrocyte-derived neurotrophic factor

P10620

17587.2

128%

3%

60S ribosomal protein L8

Q9NSE4

113719.1

121%

4%

Peptidyl-prolyl cis-trans isomerase A

O00469

84631.8

120%

2%

39S ribosomal protein L49

P20340

23577.9

100%

100%

60S ribosomal protein L3

P80723

22680

100%

33%

Endoplasmic reticulum resident protein 44

Q96AQ6

80594.2

100%

27%

40S ribosomal protein S7

P08962

25619.1

100%

25%

Prolyl 4-hydroxylase subunit alpha-2

P50281

65842

100%

25%

Transmembrane emp24 domain-containing protein 4

P62988

8559.6

100%

23%

Electron transfer flavoprotein subunit alpha

P21291

20553.8

100%

21%

Protein S100-A10

P62879

37307.1

100%

21%

40S ribosomal protein S23

Q92499

82379.9

100%

15%

Myosin-11

P05388

34251.8

100%

14%

Heterogeneous nuclear ribonucleoprotein D0

O96000

20763.2

100%

12%

Catenin beta-1

P23528

18490.7

100%

12%

Seprase

P15313

56797

100%

12%

Translocation protein SEC62

Q9UBS4

40488.7

100%

12%

Mitochondrial 2-oxoglutarate/malate carrier protein

P62917

28007.3

100%

12%

Microsomal glutathione S-transferase 1

P00403

25548.2

100%

12%

X-ray repair cross-complementing protein 6

P17813

70533.2

100%

11%

Talin-1

P61353

15787.8

100%

11%

Mannosyl-oligosaccharide glucosidase

Q00341

141368

100%

11%

Neutral cholesterol ester hydrolase 1

P0C7P4

30796.1

100%

11%

Probable ATP-dependent RNA helicase DDX17

P49368

60495.4

100%

11%

Cytochrome b-c1 complex subunit 7

Q9NQC3

129851.2

100%

10%

40S ribosomal protein S15a

Q9NX63

26136.2

100%

10%

Interleukin enhancer-binding factor 3

P84098

23451.3

100%

10%

Protein ETHE1

Q12906

95279.2

100%

10%

Coiled-coil-helix-coiled-coil-helix domain-containing protein 3

P11166

54048.7

100%

9%

Estradiol 17-beta-dehydrogenase 12

P06748

32554.9

100%

9%

Heterogeneous nuclear ribonucleoprotein M

Q99584

11464.1

100%

9%

Mitochondrial import receptor subunit TOM40 homolog

P14927

13522

100%

8%

T-complex protein 1 subunit gamma

Q9UBI6

8001.2

100%

8%

Pyruvate dehydrogenase E1 component subunit alpha

P83731

17767.9

100%

8%

Protein canopy homolog 2

Q02218

115861.5

100%

8%

Spectrin alpha chain

P61026

22526.6

100%

7%

Filamin-A

O95299

40725

100%

7%

Myosin-Ic

P22626

37406.7

100%

7%

Ras-related protein Rap-1A

Q9UBG0

166548.2

100%

7%

Succinyl-CoA ligase [GDP-forming] subunit beta

P07099

52915

100%

7%

RNA-binding protein Raly

O95182

12543.6

100%

7%

Collagen alpha-2(VI) chain

P04216

17923.4

100%

7%

Ras GTPase-activating-like protein IQGAP1

P62266

15797.7

100%

7%

Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit

beta-2

P35221

100008.6

100%

7%

V-type proton ATPase subunit B

Q13405

19186

100%

7%

Procollagen-lysine

P18859

12579.6

100%

7%

60S ribosomal protein L32

P25398

14505.5

100%

7%

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase

subunit STT3A

O00571

73198.1

100%

7%

Alpha-soluble NSF attachment protein

P00367

61359.3

100%

7%

ATPase family AAA domain-containing protein 3A

P62081

22113.3

100%

6%

Pre-B-cell leukemia transcription factor-interacting protein 1

Q15293

38866.2

100%

6%

Delta-1-pyrroline-5-carboxylate synthase

P21333

280561.4

100%

6%

Ras-related protein Rab-10

P54920

33211.3

100%

6%

ATP-dependent RNA helicase DDX3X

P62249

16435

100%

6%

DnaJ homolog subfamily B member 11

P62834

20973.7

100%

6%

Heterogeneous nuclear ribonucleoproteins A2/B1

O00264

21657.8

100%

6%

Thioredoxin domain-containing protein 5

Q16718

13450.2

100%

6%

Calmodulin

Q9Y3B3

25155.6

100%

6%

40S ribosomal protein S27-like

Q01518

51822.8

100%

6%

Citrate synthase

P35222

85442.3

100%

6%

NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit

10

Q9UKM9

32443.6

100%

6%

Epoxide hydrolase 1

P51991

39570.5

100%

6%

60S ribosomal protein L36

P04899

40425.1

100%

6%

UPF0027 protein C22orf28

P62244

14830

100%

6%

Reticulon-4

P32322

33339.6

100%

5%

Sarcoplasmic/endoplasmic reticulum calcium ATPase 1

P52815

21334.7

100%

5%

Peptidyl-prolyl cis-trans isomerase FKBP11

Q15019

41461.3

100%

5%

Histone H2A type 1-A

Q13011

35793.4

100%

5%

Gelsolin

P00558

44586.2

100%

5%

ATP synthase subunit g

O75439

54331.6

100%

5%

Thrombospondin-1

P43304

80801.7

100%

5%

Histone H1.5

P18084

87996.2

100%

5%

Plasminogen activator inhibitor 1

Q7Z7H5

25926.4

100%

5%

Matrix metalloproteinase-14

Q14554

59556.2

100%

5%

Peptidyl-prolyl cis-trans isomerase FKBP10

O60506

69559.6

100%

5%

Enoyl-CoA hydratase

Q9Y3U8

12245.9

100%

5%

Heterogeneous nuclear ribonucleoprotein A3

P62820

22663.4

100%

5%

Fumarate hydratase

Q13813

284362.5

100%

5%

2-oxoglutarate dehydrogenase

Q6P587

24826.7

100%

5%

Interleukin enhancer-binding factor 2

O75964

11421.2

100%

5%

Phosphoglycerate kinase 1

Q9Y2B0

20639.2

100%

4%

60S ribosomal protein L24

Q12931

80059.8

100%

4%

Myeloid-associated differentiation marker

P51572

27974

100%

4%

Alpha-actinin-4

Q5JRX3

117380.3

100%

4%

Ornithine aminotransferase

P36776

106422.5

100%

4%

UPF0556 protein C19orf10

O95571

27855.1

100%

4%

60S ribosomal protein L19

P61158

47341

100%

4%

NADH dehydrogenase [ubiquinone] flavoprotein 1

Q9BS26

46941.5

100%

4%

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 7

P10515

68953.3

100%

4%

40S ribosomal protein S2

Q71UM5

9470.9

100%

4%

Transmembrane emp24 domain-containing protein 7

Q12905

43035.2

100%

4%

Fumarylacetoacetate hydrolase domain-containing protein 1

P54886

87247.7

100%

4%

Reticulocalbin-1

P13073

19564.1

100%

4%

Cathepsin B

P15880

31304.6

100%

4%

Dihydrolipoyllysine-residue acetyltransferase component of

pyruvate dehydrogenase complex

P60866

13364.3

100%

4%

Aspartyl/asparaginyl beta-hydroxylase

Q96I99

46481.5

100%

3%

Catechol O-methyltransferase

O96008

37869.2

100%

3%

Protein S100-A13

P61313

24131.1

100%

3%

Alpha-aminoadipic semialdehyde dehydrogenase

Q6PIU2

45778.8

100%

3%

Vigilin

Q8NBS9

47598.7

100%

3%

Membrane-associated progesterone receptor component 1

Q14165

32213.6

100%

3%

Galectin-1

P04181

48504.3

100%

3%

Presequence protease

Q9NVI7

71324.8

100%

3%

Glutamate dehydrogenase 1

Q92896

134463.3

100%

3%

Pyruvate dehydrogenase E1 component subunit beta

Q99653

22442.4

100%

3%

Profilin-1

P54709

31492.1

100%

3%

Serine beta-lactamase-like protein LACTB

Q92520

24664.6

100%

3%

40S ribosomal protein S17

P53597

36226.9

100%

3%

3-ketoacyl-CoA thiolase

P15311

69369.8

100%

3%

Myosin light polypeptide 6

P13804

35057.6

100%

3%

Ubiquitin

O00217

23689.6

100%

3%

40S ribosomal protein S16

Q9UIJ7

25549.6

100%

3%

Microsomal glutathione S-transferase 3

P62158

16826.8

100%

3%

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 5

Q969H8

18783.3

100%

3%

Lon protease homolog

P83111

60655.1

100%

3%

Sodium/potassium-transporting ATPase subunit beta-3

Q12884

87656.8

100%

3%

Cofilin-1

P07954

54602.2

100%

3%

Ras-related protein Rab-1A

Q14697

106806.8

100%

3%

Endoplasmic reticulum-Golgi intermediate compartment protein 1

O14983

110181.8

100%

2%

Pyrroline-5-carboxylate reductase 1

Q99714

26906.1

100%

2%

Ras-related protein Rab-5C

P20020

138667.9

100%

2%

60S ribosomal protein L17

P62910

15849.8

100%

2%

ATP synthase-coupling factor 6

P39023

46079.8

100%

2%

Ras-related protein Rab-6A

Q9NYL4

22166.3

100%

2%

39S ribosomal protein L12

O14880

16505.6

100%

2%

GTP:AMP phosphotransferase mitochondrial

Q92841

72326

100%

2%

Putative cytochrome b-c1 complex subunit Rieske-like protein 1

Q96QV6

14224.9

100%

2%

Septin-2

Q9Y224

28050.7

100%

2%

Catenin alpha-1

P55809

56122

100%

2%

Transmembrane emp24 domain-containing protein 2

Q13724

91860.9

100%

1%

60S ribosomal protein L27

Q6DD88

60503.5

100%

1%

Epididymal secretory protein E1

Q96AY3

64204.3

100%

1%

Protein disulfide-isomerase A5

Q9Y6C9

33308.9

100%

1%

C-type mannose receptor 2

O15460

60863.7

100%

1%

CD63 antigen

Q53GQ0

34302.2

100%

1%

Solute carrier family 2

Q07021

31342.6

100%

1%

Apoptosis-inducing factor 1

P60660

16919.1

100%

0%

Ezrin

P35749

227197.9

100%

0%

ATP-dependent RNA helicase DDX1

P08559

43267.7

100%

0%

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit

gamma-12

O15260

30373.8

100%

0%

Tropomyosin alpha-1 chain

P16401

22566.5

100%

0%

Glycerol-3-phosphate dehydrogenase

P11177

39208.1

82%

4%

Golgi apparatus protein 1

P12110

108511.9

81%

3%

Brain acid soluble protein 1

P42765

41897.7

79%

2%

Succinyl-CoA ligase [GDP-forming] subunit alpha

O94925

73414

76%

3%

Catenin delta-1

Q9Y3I0

55175

72%

8%

Guanine nucleotide-binding protein G(i) subunit alpha-2

P55145

20243.6

71%

2%

Tripeptidyl-peptidase 1

O43707

104788.5

71%

4%

B-cell receptor-associated protein 31

P12814

102992.7

70%

4%

Carnitine O-palmitoyltransferase 1

P49419

58450.2

66%

3%

60S ribosomal protein L15

P09493

32688.7

66%

27%

Surfeit locus protein 4

P35914

34337.8

62%

8%

UDP-glucose:glycoprotein glucosyltransferase 1

P37802

22377.2

61%

4%

60S ribosomal protein L23a

P07737

15044.6

57%

7%

Calcium-binding protein p22

P08708

15540.4

57%

5%

Protein FAM3C

P30084

31367.1

57%

3%

Heterogeneous nuclear ribonucleoprotein Q

P62937

18000.9

54%

9%

Isoleucyl-tRNA synthetase

P19338

76568.5

50%

42%

Acyl-CoA dehydrogenase family member 9

P09382

14706.2

45%

28%

Malectin

P06396

85644.3

36%

2%

Delta(3

P02461

138479.2

35%

6%

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase

subunit STT3B

Q9Y490

269596.3

34%

5%

Endoglin

P62750

17684.1

24%

31%

Transgelin-2

[0261]

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0262]

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

REFERENCEOther References are Cited Throughout the Application

[0000]

1. Petersson, B. The dry mass of the pancreatic B-cells in relation to their content of secretion granules. The Histochemical Journal 1, 55-58 (1968).